Remnant Tumor Infiltrating Lymphocytes and Methods of Preparing and Using the Same

ABSTRACT

In some embodiments, methods of delivering a therapeutically effective amount of an expanded number of tumor infiltrating lymphocytes obtained from tumor remnants to a patient in need thereof, for the treatment of a cancer, are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This international application claims the benefit of priority to U.S.Provisional Application No. 62/423,750, filed Nov. 17, 2016, and U.S.Provisional Application No. 62/460,441, filed Feb. 17, 2017, theentirety of which are incorporated herein by reference.

FIELD OF THE INVENTION

Methods and compositions for expansion of tumor infiltrating lymphocytesfrom tumor remnants are disclosed in some embodiments.

BACKGROUND OF THE INVENTION

Treatment of bulky, refractory cancers using adoptive transfer of tumorinfiltrating lymphocytes (TILs) represents a powerful approach totherapy for patients with poor prognoses. Gattinoni, et al., Nat. Rev.Immunol. 2006, 6, 383-393. Adoptive T cell therapy with autologous TILsprovides up to 55% objective response rates and durable regressionin >25% of patients with metastatic melanoma. A large number of TILs arerequired for successful immunotherapy, and a robust and reliable processis needed for commercialization. This has been a challenge to achievebecause of technical, logistical, and regulatory issues with cellexpansion. IL-2-based TIL expansion followed by a “rapid expansionprocess” (REP) has become a preferred method for TIL expansion becauseof its speed and efficiency. Dudley, et al., Science 2002, 298, 850-54;Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J.Clin. Oncol. 2008, 26, 5233-39; Riddell, et al., Science 1992, 257,238-41; Dudley, et al., J. Immunother. 2003, 26, 332-42. Processes forgenerating TILs from resected tumors include the step of morcellatingthe tumor into 1-3 mm³ fragments, and expanding TILs in the presence ofinterleukin 2 (IL-2) in the pre-rapid expansion protocol (pre-REP orinitiation) step. During the pre-REP step, tumor-resident immune cellsemigrate and proliferate, and these TILs are subjected to a second REPprocess, with irradiated peripheral blood mononuclear cell (PBMC)feeders, anti-CD3 antibody (OKT-3, muromonab), and IL-2, which greatlyincreases their numbers. To date, all TIL expansion processes discardresidual tumor fragments following the pre-REP process.

Direct enzymatic digestion of resected tumors has been previouslyexplored as an alternative to pre-REP, but has been reported to yieldless TIL cultures, resulting in a decreased ability to obtain TILs thanfrom pre-REP initiation processes with IL-2. Dudley, et al., J.Immunother. 2003, 26, 332-42. For this reason, digestion has not beenfurther explored in the development of TILs as a therapy for cancer.

TILs obtained from the pre-REP and REP processes have dominated theclinical studies of TILs to date, which have offered modest clinicalresponses, and the field remains challenging, particularly in theextension of TIL therapy from melanoma to other tumor types. Goff, etal., J. Clin. Oncol. 2016, 34, 2389-97; Dudley, et al., J. Clin. Oncol.2008, 26, 5233-39; Rosenberg, et al., Clin. Cancer Res. 2011, 17,4550-57. Much focus has been placed on selection of TILs duringexpansion to either select particular subsets (such as CD8⁺ T cells) orto target driver mutations such as a mutated ERBB2IP epitope or drivermutations in the KRAS oncogene. Tran, et al., N. Engl. J. Med. 2016,375, 2255-62; Tran, et al., Science 2014, 344, 641-45. However, suchselection approaches, even if they can be developed to show efficacy inlarger clinical trials, add significantly to the duration, complexity,and cost of performing TIL therapy and limit the potential forwidespread use of TIL therapy in different types of cancers. Thus, thereis an urgent need to develop processes capable of providing TILs withimproved properties for use in cancer therapies.

The invention provides the unexpected finding that TILs with improvedproperties may be obtained from processes based on tumor remnant cells,and that such remnant TILs (rTILs) are phenotypically and functionallydistinct from normal emigrant TILs (eTILs). The use of rTILs andcombinations of rTILs and eTILs in cancer immunotherapy providessignificant advantages over prior eTIL-based therapies.

SUMMARY OF THE INVENTION

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs, and wherein the T cell exhaustion        marker is selected from the group consisting of TIM3, LAG3,        TIGIT, PD-1, CTLA-4, and combinations thereof.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, CTLA-4, and combinations        thereof, and    -   wherein the tumor tissue is selected from the group consisting        of melanoma tumor tissue, head and neck tumor tissue, breast        tumor tissue, renal tumor tissue, pancreatic tumor tissue,        glioblastoma tumor tissue, lung tumor tissue, colorectal tumor        tissue, sarcoma tumor tissue, triple negative breast tumor        tissue, cervical tumor tissue, ovarian tumor tissue, and acute        myeloid leukemia bone marrow or tumor tissue.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, CTLA-4, and combinations        thereof, and    -   wherein the irradiated feeder cells comprise irradiated        allogeneic peripheral blood mononuclear cells.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein IL-2 is present in the second cell culture medium at an        initial concentration of about 3000 IU/mL and OKT-3 antibody is        present in the second cell culture medium at an initial        concentration of about 30 ng/mL.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein at least one T cell exhaustion marker in CD8⁺ and CD4⁺ T        cells in the rTILs is reduced by at least 10% relative to the        eTILs.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the T cell exhaustion marker is a LAG3 marker in CD8⁺ T        cells, and wherein the LAG3 marker in the rTILs is reduced by at        least 2-fold relative to the eTILs.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the T cell exhaustion marker is a TIM3 marker in CD8⁺ T        cells, and wherein the LAG3 marker in the rTILs is reduced by at        least 3-fold relative to the eTILs.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the T cell exhaustion marker is a TIM3 marker in CD4⁺ T        cells, and wherein the LAG3 marker in the rTILs is reduced by at        least 2-fold relative to the eTILs.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the TIM3 marker and the LAG3 marker in the rTILs are        undetectable by flow cytometry.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein CD56⁺ expression in the rTILs is reduced by at least        3-fold relative to CD56⁺ expression in the eTILs.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein CD69⁺ expression in the rTILs is increased by at least        2-fold relative to CD69⁺ expression in the eTILs.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the digest mixture comprises deoxyribonuclease,        collagenase, and hyaluronidase.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the first cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the second cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof.

In an embodiment, the invention includes a method of treating a cancerin a patient in need of such treatment, wherein the treatment comprisesdelivering a therapeutically effective amount of rTILs to a patient,wherein the rTILs are prepared according a method comprising the stepsof:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the second cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof.

In an embodiment, the invention includes a method treating a cancer in apatient in need of such treatment, the method comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture;    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and wherein the second cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof,    -   (g) treating the patient with a non-myeloablative        lymphodepletion regimen prior to administering the rTILs to the        patient;    -   (h) administering a therapeutically effective amount of rTILs to        the patient; and    -   (i) treating the patient with a high-dose IL-2 regimen starting        on the day after administration of the rTILs to the patient.

In an embodiment, the invention includes a method treating a cancer in apatient in need of such treatment, the method comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture;    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the second cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof,    -   (g) treating the patient with a non-myeloablative        lymphodepletion regimen prior to administering the rTILs to the        patient;    -   (h) administering a therapeutically effective amount of rTILs to        the patient, wherein a therapeutically effective amount of eTILs        are simultaneously administered to the patient in a mixture with        the rTILs; and    -   (i) treating the patient with a high-dose IL-2 regimen starting        on the day after administration of the rTILs to the patient.

In an embodiment, the invention includes a method treating a cancer in apatient in need of such treatment, the method comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture;    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the second cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof,    -   (g) treating the patient with a non-myeloablative        lymphodepletion regimen prior to administering the rTILs to the        patient;    -   (h) administering a therapeutically effective amount of rTILs to        the patient, wherein a therapeutically effective amount of eTILs        are simultaneously administered to the patient in a mixture with        the rTILs; and    -   (i) treating the patient with a high-dose IL-2 regimen starting        on the day after administration of the rTILs to the patient,    -   wherein the cancer is selected from the group consisting of        melanoma, double-refractory melanoma, ovarian cancer, cervical        cancer, lung cancer, bladder cancer, breast cancer, head and        neck cancer, renal cell carcinoma, acute myeloid leukemia,        colorectal cancer, sarcoma, non-small cell lung cancer (NSCLC),        and triple negative breast cancer.

In an embodiment, the invention includes a method treating a cancer in apatient in need of such treatment, the method comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture;    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the second cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof,    -   (g) treating the patient with a non-myeloablative        lymphodepletion regimen prior to administering the rTILs to the        patient, wherein the non-myeloablative lymphodepletion regimen        comprises the steps of administration of cyclophosphamide at a        dose of 60 mg/m²/day for two days followed by administration of        fludarabine at a dose of 25 mg/m²/day for five days;    -   (h) administering a therapeutically effective amount of rTILs to        the patient, wherein a therapeutically effective amount of eTILs        are simultaneously administered to the patient in a mixture with        the rTILs; and    -   (i) treating the patient with a high-dose IL-2 regimen starting        on the day after administration of the rTILs to the patient.

In an embodiment, the invention includes a method treating a cancer in apatient in need of such treatment, the method comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture;    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);

wherein the rTILs express reduced levels of a T cell exhaustion markerrelative to the eTILs, wherein the T cell exhaustion marker is selectedfrom the group consisting of TIM3, LAG3, TIGIT, PD-1, and combinationsthereof, and wherein the second cell culture medium further comprises acytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21,and combinations thereof,

-   -   (g) treating the patient with a non-myeloablative        lymphodepletion regimen prior to administering the rTILs to the        patient;    -   (h) administering a therapeutically effective amount of rTILs to        the patient, wherein a therapeutically effective amount of eTILs        are simultaneously administered to the patient in a mixture with        the rTILs; and    -   (i) treating the patient with a high-dose IL-2 regimen starting        on the day after administration of the rTILs to the patient,    -   wherein the high-dose IL-2 regimen comprises 600,000 or 720,000        IU/kg of aldesleukin, or a biosimilar or variant thereof,        administered as a 15-minute bolus intravenous infusion every        eight hours until tolerance.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs, and wherein the T cell exhaustion        marker is selected from the group consisting of TIM3, LAG3,        TIGIT, PD-1, CTLA-4, and combinations thereof.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, CTLA-4, and combinations        thereof, and    -   wherein the tumor tissue is selected from the group consisting        of melanoma tumor tissue, head and neck tumor tissue, breast        tumor tissue, renal tumor tissue, pancreatic tumor tissue,        glioblastoma tumor tissue, lung tumor tissue, colorectal tumor        tissue, sarcoma tumor tissue, triple negative breast tumor        tissue, cervical tumor tissue, ovarian tumor tissue, and acute        myeloid leukemia bone marrow or tumor tissue.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, CTLA-4, and combinations        thereof, and    -   wherein the irradiated feeder cells comprise irradiated        allogeneic peripheral blood mononuclear cells.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein IL-2 is present in the second cell culture medium at an        initial concentration of about 3000 IU/mL and OKT-3 antibody is        present in the second cell culture medium at an initial        concentration of about 30 ng/mL.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein at least one T cell exhaustion marker in CD8⁺ and CD4⁺ T        cells in the rTILs is reduced by at least 10% relative to the        eTILs.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the T cell exhaustion marker is a LAG3 marker in CD8⁺ T        cells, and wherein the LAG3 marker in the rTILs is reduced by at        least 2-fold relative to the eTILs.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the T cell exhaustion marker is a TIM3 marker in CD8⁺ T        cells, and wherein the LAG3 marker in the rTILs is reduced by at        least 3-fold relative to the eTILs.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the T cell exhaustion marker is a TIM3 marker in CD4⁺ T        cells, and wherein the LAG3 marker in the rTILs is reduced by at        least 2-fold relative to the eTILs.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the TIM3 marker and the LAG3 marker in the rTILs are        undetectable by flow cytometry.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein CD56⁺ expression in the rTILs is reduced by at least        3-fold relative to CD56⁺ expression in the eTILs.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein CD69⁺ expression in the rTILs is increased by at least        2-fold relative to CD69⁺ expression in the eTILs.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the digest mixture comprises deoxyribonuclease,        collagenase, and hyaluronidase.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the first cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof.

In an embodiment, the invention includes a method for preparing remnanttumor infiltrating lymphocytes (rTILs) for adoptive T cell therapy, themethod comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the second cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof.

In an embodiment, the invention includes a method of treating a cancerin a patient in need of such treatment, wherein the treatment comprisesdelivering a therapeutically effective amount of rTILs to a patient,wherein the rTILs are prepared according a method comprising the stepsof:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the second cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof.

In an embodiment, the invention includes a method treating a cancer in apatient in need of such treatment, the method comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture;    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the second cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof,    -   (g) treating the patient with a non-myeloablative        lymphodepletion regimen prior to administering the rTILs to the        patient;    -   (h) administering a therapeutically effective amount of rTILs to        the patient; and    -   (i) treating the patient with a high-dose IL-2 regimen starting        on the day after administration of the rTILs to the patient.

In an embodiment, the invention includes a method treating a cancer in apatient in need of such treatment, the method comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture;    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the second cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof,    -   (g) treating the patient with a non-myeloablative        lymphodepletion regimen prior to administering the rTILs to the        patient;    -   (h) administering a therapeutically effective amount of rTILs to        the patient, wherein a therapeutically effective amount of eTILs        are simultaneously administered to the patient in a mixture with        the rTILs; and    -   (i) treating the patient with a high-dose IL-2 regimen starting        on the day after administration of the rTILs to the patient.

In an embodiment, the invention includes a method treating a cancer in apatient in need of such treatment, the method comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture;    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the second cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof,    -   (g) treating the patient with a non-myeloablative        lymphodepletion regimen prior to administering the rTILs to the        patient;    -   (h) administering a therapeutically effective amount of rTILs to        the patient, wherein a therapeutically effective amount of eTILs        are simultaneously administered to the patient in a mixture with        the rTILs; and    -   (i) treating the patient with a high-dose IL-2 regimen starting        on the day after administration of the rTILs to the patient,    -   wherein the cancer is selected from the group consisting of        melanoma, double-refractory melanoma, ovarian cancer, cervical        cancer, lung cancer, bladder cancer, breast cancer, head and        neck cancer, renal cell carcinoma, acute myeloid leukemia,        colorectal cancer, sarcoma, non-small cell lung cancer (NSCLC),        and triple negative breast cancer.

In an embodiment, the invention includes a method treating a cancer in apatient in need of such treatment, the method comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture;    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the second cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof,    -   (g) treating the patient with a non-myeloablative        lymphodepletion regimen prior to administering the rTILs to the        patient, wherein the non-myeloablative lymphodepletion regimen        comprises the steps of administration of cyclophosphamide at a        dose of 60 mg/m²/day for two days followed by administration of        fludarabine at a dose of 25 mg/m²/day for five days;    -   (h) administering a therapeutically effective amount of rTILs to        the patient, wherein a therapeutically effective amount of eTILs        are simultaneously administered to the patient in a mixture with        the rTILs; and    -   (i) treating the patient with a high-dose IL-2 regimen starting        on the day after administration of the rTILs to the patient.

In an embodiment, the invention includes a method treating a cancer in apatient in need of such treatment, the method comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture;    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the second cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof,    -   (g) treating the patient with a non-myeloablative        lymphodepletion regimen prior to administering the rTILs to the        patient;    -   (h) administering a therapeutically effective amount of rTILs to        the patient, wherein a therapeutically effective amount of eTILs        are simultaneously administered to the patient in a mixture with        the rTILs; and    -   (i) treating the patient with a high-dose IL-2 regimen starting        on the day after administration of the rTILs to the patient,    -   wherein the high-dose IL-2 regimen comprises 600,000 or 720,000        IU/kg of aldesleukin, or a biosimilar or variant thereof,        administered as a 15-minute bolus intravenous infusion every        eight hours until tolerance.

In an embodiment, the invention includes a process for generating anexpanded number of tumor remnant cells that include tumor infiltratinglymphocytes (TILs) from a patient for adoptive T cell therapy. In someembodiments, the process of the invention may include the step ofobtaining tumor tissue from the patient, wherein the tumor tissuecomprises TILs. In some embodiments, the process of the invention mayinclude the step of fragmenting the tumor tissue. In some embodiments,the process of the invention may include the step of treating the tumortissue in a gas permeable container with cell culture media andinterleukin 2 (IL-2) and other T cell growth factors or agonisticantibodies to provide tumor remnants and an expanded number of TILs. Insome embodiments, the process of the invention may include the step ofremoving the expanded number of TILs. In some embodiments, the processof the invention may include the step of enzymatically digesting thetumor remnants into tumor remnant cells. In some embodiments, theprocess of the invention may include the step of treating the tumorremnant cells with cell culture media, irradiated feeder cells, anti-CD3monoclonal antibody (muromonab or OKT-3), and IL-2 to provide theexpanded number of tumor remnant cells. In some embodiments, the tumorremnant cells prepared according to the processes of the invention mayinclude TILs that express reduced levels of at least one marker selectedfrom the group consisting of TIM3, LAG3, PD-1, and combinations thereof.In some embodiments, the tumor tissue may be selected from the groupconsisting of melanoma tumor tissue, head and neck tumor tissue, breasttumor tissue, renal tumor tissue, pancreatic tumor tissue, lung tumortissue, and colorectal tumor tissue.

In an embodiment, the invention may include method of treating a tumorin a patient in need of such treatment. In some embodiments, thetreatment may include delivering a therapeutically effective amount ofan expanded number of tumor remnant cells to the patient, wherein theexpanded number of tumor remnant cells may be prepared according to anyprocess described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings.

FIG. 1 illustrates an exemplary diagram of the tumor digestion solutionpreparation.

FIG. 2 illustrates an exemplary flow-through diagram of the tumordigestion procedure. In this example, two digestion methods areperformed simultaneously and are seeded separately as a part of twodistinct pre-REPs designed to compare the efficacy of each digestionmethod.

FIG. 3 illustrates differential phenotypic expression of key markers ineTILs and rTILs.

FIG. 4 illustrates studies of eTIL and rTIL by2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG)(A) and Mitotracker (B) to assess metabolic capacity prior to rapidexpansion.

FIG. 5 shows the results of experiments wherein eTIL and rTIL werestimulated with CD3/CD28/4-1BB beads with brefeldin A overnight for CD4⁺and CD8⁺ T cells. PMA and ionomycin was added for 4-5 hours.Interferon-γ was assessed by intracellular flow cytometric analysis(n=3).

FIG. 6 illustrates results showing that (A) rTIL expand and (B) remainphenotypically distinct from eTIL during rapid expansion.

FIG. 7 illustrates an exemplary process for treating a patient usingrTILs of the invention.

FIG. 8 illustrates an exemplary timeline of the process for treating apatient using rTILs of the invention.

FIG. 9 illustrates the diversity of the TCRvβ repetoire (i.e., thediversity score) in eTIL and rTIL.

FIG. 10 illustrates the percent of shared CDR3s in eTIL and rTIL.

FIG. 11 illustrates cell proliferation analyses in triple negativebreast carcinoma, colorectal carcinoma, lung carcinoma, renal carcinoma,and melanoma where eTIL from either the CD4+ or CD8+ population in allfive tumors demonstrated an enhancement in the proliferative capacityupon co-culture with rTIL with anti-CD3 antibody as demonstrated by ashift (or dye dilution) in the Cell Trace dye, when compared to eTILalone. The red represents the eTIL and the blue represents the eTIL whenco-cultured with the rTIL.

FIG. 12 illustrates a heat map prepared from a Nanostring analysis,which shows that the gene expression profile for eTIL and rTIL issignificantly different.

FIG. 13 illustrates a graph prepared from a Nanostring analysis, whichshows that several genes are significantly upregulated or downregulatedin the rTIL as compared to the eTIL.

FIG. 14 illustrates a clonotype graph showing the top 50 shared CDR3sbetween eTIL and rTIL (for three eTIL/rTIL pairs) from ovariancarcinoma.

FIG. 15 illustrates a clonotype graph showing the top 50 shared CDR3sbetween eTIL and rTIL (for three eTIL/rTIL pairs) from renal carcinoma.

FIG. 16 illustrates a clonotype graph showing the top 50 shared CDR3sbetween eTIL and rTIL (for three eTIL/rTIL pairs) from triple negativebreast carcinoma.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1 is the amino acid sequence of the heavy chain of muromonab.

SEQ ID NO:2 is the amino acid sequence of the light chain of muromonab.

SEQ ID NO:3 is the amino acid sequence of a recombinant human IL-2protein.

SEQ ID NO:4 is the amino acid sequence of aldesleukin.

SEQ ID NO:5 is the amino acid sequence of a recombinant human IL-4protein.

SEQ ID NO:6 is the amino acid sequence of a recombinant human IL-7protein.

SEQ ID NO:7 is the amino acid sequence of a recombinant human IL-15protein.

SEQ ID NO:8 is the amino acid sequence of a recombinant human IL-21protein.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are incorporated by reference in their entireties.

Definitions

The terms “exhausted phenotype” and “exhaustion marker” refer to cellsurface markers characteristic of T cell exhaustion in response tochronic T cell receptor (TCR) stimulation by antigen. T cells exhibitingan exhausted phenotype express inhibitory receptors, such as T cellimmunoglobulin and mucin-domain containing-3 (TIM3 or TIM-3),lymphocyte-activation gene 3 (LAG3 or LAG-3), T cell immunoreceptor withimmunoglobulin and ITIM domains (TIGIT), and programmed cell deathprotein 1 (PD-1), and lack effector cytokine production and the abilityto mount an effective immune response. Exhaustion in T cells isdescribed in Yi, et al., Immunology 2010, 129, 474-81, the disclosure ofwhich is incorporated by reference herein.

The terms “co-administration,” “co-administering,” “administered incombination with,” “administering in combination with,” “simultaneous,”and “concurrent,” as used herein, encompass administration of two ormore active pharmaceutical ingredients to a human subject so that bothactive pharmaceutical ingredients and/or their metabolites are presentin the human subject at the same time. Co-administration includessimultaneous administration in separate compositions, administration atdifferent times in separate compositions, or administration in acomposition in which two or more active pharmaceutical ingredients arepresent. Simultaneous administration in separate compositions andadministration in a composition in which both agents are present is alsoencompassed in the methods of the invention.

The term “in vivo” refers to an event that takes place in a subject'sbody.

The term “in vitro” refers to an event that takes places outside of asubject's body. In vitro assays encompass cell-based assays in whichcells alive or dead are employed and may also encompass a cell-freeassay in which no intact cells are employed.

The term “antigen” refers to a substance that induces an immuneresponse. In some embodiments, an antigen is a molecule capable of beingbound by an antibody or a TCR if presented by major histocompatibilitycomplex (MHC) molecules. The term “antigen”, as used herein, alsoencompasses T cell epitopes. An antigen is additionally capable of beingrecognized by the immune system. In some embodiments, an antigen iscapable of inducing a humoral immune response or a cellular immuneresponse leading to the activation of B lymphocytes and/or Tlymphocytes. In some cases, this may require that the antigen containsor is linked to a Th cell epitope. An antigen can also have one or moreepitopes (e.g., B- and T-epitopes). In some embodiments, an antigen willpreferably react, typically in a highly specific and selective manner,with its corresponding antibody or TCR and not with the multitude ofother antibodies or TCRs which may be induced by other antigens.

The term “effective amount” or “therapeutically effective amount” refersto that amount of a compound or combination of compounds as describedherein that is sufficient to effect the intended application including,but not limited to, disease treatment. A therapeutically effectiveamount may vary depending upon the intended application (in vitro or invivo), or the human subject and disease condition being treated (e.g.,the weight, age and gender of the subject), the severity of the diseasecondition, the manner of administration, etc. which can readily bedetermined by one of ordinary skill in the art. The term also applies toa dose that will induce a particular response in target cells (e.g., thereduction of platelet adhesion and/or cell migration). The specific dosewill vary depending on the particular compounds chosen, the dosingregimen to be followed, whether the compound is administered incombination with other compounds, timing of administration, the tissueto which it is administered, and the physical delivery system in whichthe compound is carried. A therapeutically effective amount may be “ananti-tumor effective amount” and/or a “tumor-inhibiting effectiveamount,” which may be the precise amount of the compositions of thepresent invention to be administered can be determined by a physicianwith consideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the cytotoxic lymphocytes or rTILs described herein may beadministered at a dosage of 10⁴ to 10¹¹ cells/kg body weight (e.g., 10⁵to 10⁶, 10⁵ to 10¹⁰, 10⁵ to 10¹¹, 10⁶ to 10¹⁰, 10⁶ to 10¹¹, 10⁷ to 10¹¹,10⁷ to 10¹⁰, 10⁸ to 10¹¹, 10⁸ to 10¹⁰, 10⁹ to 10¹¹, or 10⁹ to 10¹⁰cells/kg body weight), including all integer values within those ranges.Cytotoxic lymphocyte or rTIL compositions may also be administeredmultiple times at these dosages. The cytotoxic lymphocytes or rTILs canbe administered by using infusion techniques that are commonly known inimmunotherapy (see, e.g., Rosenberg et al., N. Eng. J. Med. 319: 1676,1988). The optimal dosage and treatment regime for a particular patientcan readily be determined by one skilled in the art of medicine bymonitoring the patient for signs of disease and adjusting the treatmentaccordingly.

A “therapeutic effect” as that term is used herein, encompasses atherapeutic benefit and/or a prophylactic benefit in a human subject. Aprophylactic effect includes delaying or eliminating the appearance of adisease or condition, delaying or eliminating the onset of symptoms of adisease or condition, slowing, halting, or reversing the progression ofa disease or condition, or any combination thereof.

“Pharmaceutically acceptable carrier” or “pharmaceutically acceptableexcipient” is intended to include any and all solvents, dispersionmedia, coatings, antibacterial and antifungal agents, isotonic andabsorption delaying agents, and inert ingredients. The use of suchpharmaceutically acceptable carriers or pharmaceutically acceptableexcipients for active pharmaceutical ingredients is well known in theart. Except insofar as any conventional pharmaceutically acceptablecarrier or pharmaceutically acceptable excipient is incompatible withthe active pharmaceutical ingredient, its use in the therapeuticcompositions of the invention is contemplated. Additional activepharmaceutical ingredients, such as other drugs, can also beincorporated into the described compositions and methods.

The terms “treatment”, “treating”, “treat”, and the like, refer toobtaining a desired pharmacologic and/or physiologic effect. The effectmay be prophylactic in terms of completely or partially preventing adisease or symptom thereof and/or may be therapeutic in terms of apartial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment”, as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its developmentor progression; and (c) relieving the disease, i.e., causing regressionof the disease and/or relieving one or more disease symptoms.“Treatment” is also meant to encompass delivery of an agent in order toprovide for a pharmacologic effect, even in the absence of a disease orcondition. For example, “treatment” encompasses delivery of acomposition that can elicit an immune response or confer immunity in theabsence of a disease condition, e.g., in the case of a vaccine.

The term “heterologous” when used with reference to portions of anucleic acid or protein indicates that the nucleic acid or proteincomprises two or more subsequences that are not found in the samerelationship to each other in nature. For instance, the nucleic acid istypically recombinantly produced, having two or more sequences fromunrelated genes arranged to make a new functional nucleic acid, e.g., apromoter from one source and a coding region from another source, orcoding regions from different sources. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

The term “rapid expansion” means an increase in the number ofantigen-specific TILs of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-,or 9-fold) over a period of a week, more preferably at least about10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a periodof a week, or most preferably at least about 100-fold over a period of aweek. A number of rapid expansion protocols are outlined herein.

By “tumor infiltrating lymphocytes” or “TILs” herein is meant apopulation of cells originally obtained as white blood cells that haveleft the bloodstream of a subject and migrated into a tumor. TILsinclude, but are not limited to, CD8⁺ cytotoxic T cells (lymphocytes),Th1 and Th17 CD4⁺ T cells, natural killer cells, dendritic cells and M1macrophages. TILs include both primary and secondary TILs. “PrimaryTILs” are those that are obtained from patient tissue samples asoutlined herein (sometimes referred to as “freshly harvested”), and“secondary TILs” are any TIL cell populations that have been expanded orproliferated as discussed herein, including, but not limited to bulkTILs and expanded TILs (“REP TILs” or “post-REP TILs”). In certainembodiments, the term “Primary TILs” may include rTILs and mixtures ofeTILs and rTILs.

By “population of cells” (including TILs) herein is meant a number ofcells that share common traits. In general, populations generally rangefrom 1×10⁶ to 1×10¹⁰ in number, with different TIL populationscomprising different numbers. For example, initial growth of primaryTILs in the presence of IL-2 results in a population of bulk TILs ofroughly 1×10⁸ cells. REP expansion is generally done to providepopulations of 1.5×10⁹ to 1.5×10¹⁰ cells for infusion.

The terms “peripheral blood mononuclear cells” and “PBMCs” refers to aperipheral blood cell having a round nucleus, including lymphocytes(such as T cells, B cells, and NK cells) and monocytes. Preferably, theperipheral blood mononuclear cells are irradiated allogeneic peripheralblood mononuclear cells. PBMCs are a type of antigen-presenting cell.

By “cryopreserved TILs” herein is meant that TILs, either primary, bulk,or expanded (REP TILs), are treated and stored in the range of about−150° C. to −60° C. General methods for cryopreservation are alsodescribed elsewhere herein. For clarity, “cryopreserved TILs” aredistinguishable from frozen tissue samples which may be used as a sourceof primary TILs including rTILs.

By “thawed cryopreserved TILs” herein is meant a population of TILs(such as rTILs) that was previously cryopreserved and then treated toreturn to room temperature or higher, including but not limited to cellculture temperatures or temperatures wherein TILs may be administered toa patient.

The terms “sequence identity,” “percent identity,” and “sequence percentidentity” in the context of two or more nucleic acids or polypeptides,refer to two or more sequences or subsequences that are the same or havea specified percentage of nucleotides or amino acid residues that arethe same, when compared and aligned (introducing gaps, if necessary) formaximum correspondence, not considering any conservative amino acidsubstitutions as part of the sequence identity. The percent identity canbe measured using sequence comparison software or algorithms or byvisual inspection. Various algorithms and software are known in the artthat can be used to obtain alignments of amino acid or nucleotidesequences. Suitable programs to determine percent sequence identityinclude for example the BLAST suite of programs available from the U.S.Government's National Center for Biotechnology Information BLAST website. Comparisons between two sequences can be carried using either theBLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acidsequences, while BLASTP is used to compare amino acid sequences. ALIGN,ALIGN-2 (Genentech, South San Francisco, Calif.) or MegAlign, availablefrom DNASTAR, are additional publicly available software programs thatcan be used to align sequences. One skilled in the art can determineappropriate parameters for maximal alignment by particular alignmentsoftware. In certain embodiments, the default parameters of thealignment software are used.

The term “conservative amino acid substitutions” means amino acidsequence modifications which do not abrogate the binding of the antibodyto the antigen. Conservative amino acid substitutions include thesubstitution of an amino acid in one class by an amino acid of the sameclass, where a class is defined by common physicochemical amino acidside chain properties and high substitution frequencies in homologousproteins found in nature, as determined, for example, by a standardDayhoff frequency exchange matrix or BLOSUM matrix. Six general classesof amino acid side chains have been categorized and include: Class I(Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gln,Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); and ClassVI (Phe, Tyr, Trp). For example, substitution of an Asp for anotherclass III residue such as Asn, Gln, or Glu, is a conservativesubstitution. Thus, a predicted nonessential amino acid residue in aprotein is preferably replaced with another amino acid residue from thesame class. Methods of identifying amino acid conservative substitutionswhich do not eliminate antigen binding are well-known in the art (see,e.g., Brummell, et al., Biochemistry 1993, 32, 1180-1187; Kobayashi, etal., Protein Eng. 1999, 12, 879-884 (1999); and Burks, et al., Proc.Natl. Acad. Sci. USA 1997, 94, 412-417).

“Pegylation” refers to a modified antibody or fusion protein, or afragment thereof, that typically is reacted with polyethylene glycol(PEG), such as a reactive ester or aldehyde derivative of PEG, underconditions in which one or more PEG groups become attached to theantibody or antibody fragment. Pegylation may, for example, increase thebiological (e.g., serum) half life of the antibody. Preferably, thepegylation is carried out via an acylation reaction or an alkylationreaction with a reactive PEG molecule (or an analogous reactivewater-soluble polymer). As used herein, the term “polyethylene glycol”is intended to encompass any of the forms of PEG that have been used toderivatize other proteins, such as mono (C₁-C₁₀) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. Theprotein or antibody to be pegylated may be an aglycosylated protein orantibody. Methods for pegylation are known in the art and can be appliedto the antibodies of the invention, as described for example in EuropeanPatent Nos. EP 0154316 and EP 0401384 and U.S. Pat. No. 5,824,778, thedisclosures of each of which are incorporated by reference herein.

The term “OKT-3” (also referred to herein as “OKT3”) refers to amonoclonal antibody or biosimilar or variant thereof, including human,humanized, chimeric, or murine antibodies, directed against the CD3receptor in the T cell antigen receptor of mature T cells, and includescommercially-available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure,Miltenyi Biotech, Inc., San Diego, Calif., USA) and muromonab orvariants, conservative amino acid substitutions, glycoforms, orbiosimilars thereof. The amino acid sequences of the heavy and lightchains of muromonab are given in Table 1 (SEQ ID NO:1 and SEQ ID NO:2).A hybridoma capable of producing OKT-3 is deposited with the AmericanType Culture Collection and assigned the ATCC accession number CRL 8001.A hybridoma capable of producing OKT-3 is also deposited with EuropeanCollection of Authenticated Cell Cultures (ECACC) and assigned CatalogueNo. 86022706. Anti-CD3 antibodies also include the UHCT1 clone(commercially available from BioLegend, San Diego, Calif., USA), alsoknown as T3 and CD3ε.

TABLE 1 Amino acid sequences of muromonab. IdentifierSequence (One-Letter Amino Acid Symbols)  SEQ ID NO: 1QVQLQQSGAE LARPGASVEM SCHASGYTFT RYTMHWVEQR PGQGLEWIGY INPSRGYTNY 60 MuromonabNQHFEDKATL TTDESSSTAY MQLSSLTSED SAVYYCARYY DDHYCLDYWG QGTTLTVSSA 120 heavy chainETTAPSVYPL APVCGGTTGS SVTLGCLVEG YFPEPVTLTW NSGSLSSGVH TFPAVLQSDL 180 YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRP ESCDETHTCP PCPAPELLGG 240 PSVFLEPPEP EDTLMISRTP EVTCVVVDVS HEDPEVEFNW YVDGVEVHNA ETKPREEQYN 300 STYRVVSVLT VLHQDWLNGE EYECKVSNKA LPAPIEKTIS KARGQPREPQ VYTLPPSRDE 360 LTENQVSLTC LVEGFYPSDI AVEWESNGQP ENNYETTPPV LDSDGSFFLY SELTVDESRW 420 QQGNVFSCSV MHEALHNHYT QESLSLSPGK 450  SEQ ID NO: 2QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMNWYQQESG TSPERWIYDT SKLASGVPAH 60 MuromonabFRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPFTFGSG TELEINRADT APTVSIFPPS 120 light chainSEQLTSGGAS VVCFLNNFYP KDINVKWEID GSERQNGVLN SWTDQDSEDS TYSMSSTLTL 180 TEDEYERHNS YTCEATHETS TSPIVESENR NEC 213 

The term “IL-2” (also referred to herein as “IL2”) refers to the T cellgrowth factor known as interleukin-2, and includes all forms of IL-2including human and mammalian forms, conservative amino acidsubstitutions, glycoforms, biosimilars, and variants thereof. IL-2 isdescribed, e.g., in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek,Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which areincorporated by reference herein. The amino acid sequence of recombinanthuman IL-2 suitable for use in the invention is given in Table 2 (SEQ IDNO:3). For example, the term IL-2 encompasses human, recombinant formsof IL-2 such as aldesleukin (PROLEUKIN, available commercially frommultiple suppliers in 22 million IU per single use vials), as well asthe form of recombinant IL-2 commercially supplied by CellGenix, Inc.,Portsmouth, N.H., USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd.,East Brunswick, N.J., USA (Cat. No. CYT-209-b) and other commercialequivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125human IL-2) is a nonglycosylated human recombinant form of IL-2 with amolecular weight of approximately 15 kDa. The amino acid sequence ofaldesleukin suitable for use in the invention is given in Table 2 (SEQID NO:4). The term IL-2 also encompasses pegylated forms of IL-2, asdescribed herein, including the pegylated IL2 prodrug NKTR-214,available from Nektar Therapeutics, South San Francisco, Calif., USA.NKTR-214 and pegylated IL-2 suitable for use in the invention isdescribed in U.S. Patent Application Publication No. US 2014/0328791 A1and International Patent Application Publication No. WO 2012/065086 A1,the disclosures of which are incorporated by reference herein.Alternative forms of conjugated IL-2 suitable for use in the inventionare described in U.S. Pat. Nos. 4,766,106, 5,206,344, 5,089,261 and4902,502, the disclosures of which are incorporated by reference herein.Formulations of IL-2 suitable for use in the invention are described inU.S. Pat. No. 6,706,289, the disclosure of which is incorporated byreference herein.

TABLE 2 Amino acid sequences of interleukins. IdentifierSequence (One-Letter Amino Acid Symbols)  SEQ ID NO: 3MAPTSSSTKK TQLQLEHLLL DLQMILNGIN NYENPKLTRM LTFKEYMPKK ATELKHLQCL 60 recombinantEEELKPLEEV LNLAQSKNFH LRPRDLISNI NVIVLELKGS ETTFMCEYAD ETATIVEFLN 120 human IL-2 RWITFCQSII STLT 134  (rhIL-2) SEQ ID NO: 4PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPELTRMLT FKFYMPKKAT ELKHLQCLEE 60 AldesleukinELKPLEEVLN LAQSENFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW 120 ITFSQSIIST LT 132  SEQ ID NO: 5MHECDITLQE IIKTLNSLTE QKTLCTELTV TDIFAASENT TEKETFCRAA TVLRQFYSHH 60 recombinantEXDTRCLGAT AQQFHRHEQL IRFLERLDRN LWGLAGLNSC PVKEANQSTL ENFLERLKTI 120 human IL-4 MREHYSECSS 130  (rhIL-4) SEQ ID NO: 6MDCDIEGEDG EQYESVLMVS IDQLLDSMKE IGSNCLNNEF NFFERHICDA NKEGMFLFRA 60 recombinantARKLRQFLKM NSTGDFDLHL LEVSEGTTIL LNCTGQVKGR KPAALGEAQP THSLEENKSL 120 human IL-7 KEQKKLNDLC FLERLLQEIK TCWNKILMGT KEH 153  (rhIL-7)SEQ ID NO: 7MNWVNVISDL KKIEDLIQSM HIDATLYTES DVHPSCEVTA MECELLELQV ISLESGDASI 60 recombinant HDTVENLIIL ANNSLSSNGN VTESGCKECE ELEEKNIKEF LQSFVHIVQM FINTS115  human IL-15 (rhIL-15) SEQ ID NO: 8MQDRHMIRMR QLIDIVDQLX NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ KAQLKSANTG 60 recombinantNNERIINVSI KELEREPPST NAGRRQKHRL TCPSCDSYEK KPPEEFLERF ESLLQHMIHQ 120 human IL-21 HLSSRTHGSE DS 132  (rhIL-21)

The term “IL-4” (also referred to herein as “IL4”) refers to thecytokine known as interleukin 4, which is produced by Th2 T cells and byeosinophils, basophils, and mast cells. IL-4 regulates thedifferentiation of naïve helper T cells (Th0 cells) to Th2 T cells.Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation byIL-4, Th2 T cells subsequently produce additional IL-4 in a positivefeedback loop. IL-4 also stimulates B cell proliferation and class IIMEW expression, and induces class switching to IgE and IgG₁ expressionfrom B cells. Recombinant human IL-4 suitable for use in the inventionis commercially available from multiple suppliers, includingProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No.CYT-211) and ThermoFisher Scientific, Inc., Waltham, Mass., USA (humanIL-4 recombinant protein, Cat. No. Gibco CTP0043). The amino acidsequence of recombinant human IL-4 suitable for use in the invention isgiven in Table 2 (SEQ ID NO:5).

The term “IL-7” (also referred to herein as “IL7”) refers to aglycosylated tissue-derived cytokine known as interleukin 7, which maybe obtained from stromal and epithelial cells, as well as from dendriticcells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate thedevelopment of T cells. IL-7 binds to the IL-7 receptor, a heterodimerconsisting of IIL-7 receptor alpha and common gamma chain receptor,which in a series of signals important for T cell development within thethymus and survival within the periphery. Recombinant human IL-4suitable for use in the invention is commercially available frommultiple suppliers, including ProSpec-Tany TechnoGene Ltd., EastBrunswick, N.J., USA (Cat. No. CYT-254) and ThermoFisher Scientific,Inc., Waltham, Mass., USA (human IL-7 recombinant protein, Cat. No.Gibco PHC0071). The amino acid sequence of recombinant human IL-7suitable for use in the invention is given in Table 2 (SEQ ID NO:6).

The term “IL-15” (also referred to herein as “IL15”) refers to the Tcell growth factor known as interleukin-15, and includes all forms ofIL-2 including human and mammalian forms, conservative amino acidsubstitutions, glycoforms, biosimilars, and variants thereof. IL-15 isdescribed, e.g., in Fehniger and Caligiuri, Blood 2001, 97, 14-32, thedisclosure of which is incorporated by reference herein. IL-15 shares βand γ signaling receptor subunits with IL-2. Recombinant human IL-15 isa single, non-glycosylated polypeptide chain containing 114 amino acids(and an N-terminal methionine) with a molecular mass of 12.8 kDa.Recombinant human IL-15 is commercially available from multiplesuppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J.,USA (Cat. No. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham,Mass., USA (human IL-15 recombinant protein, Cat. No. 34-8159-82). Theamino acid sequence of recombinant human IL-15 suitable for use in theinvention is given in Table 2 (SEQ ID NO:7).

The term “IL-21” (also referred to herein as “IL21”) refers to thepleiotropic cytokine protein known as interleukin-21, and includes allforms of IL-21 including human and mammalian forms, conservative aminoacid substitutions, glycoforms, biosimilars, and variants thereof. IL-21is described, e.g., in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014,13, 379-95, the disclosure of which is incorporated by reference herein.IL-21 is primarily produced by natural killer T cells and activatedhuman CD4⁺ T cells. Recombinant human IL-21 is a single,non-glycosylated polypeptide chain containing 132 amino acids with amolecular mass of 15.4 kDa. Recombinant human IL-21 is commerciallyavailable from multiple suppliers, including ProSpec-Tany TechnoGeneLtd., East Brunswick, N.J., USA (Cat. No. CYT-408-b) and ThermoFisherScientific, Inc., Waltham, Mass., USA (human IL-21 recombinant protein,Cat. No. 14-8219-80). The amino acid sequence of recombinant human IL-21suitable for use in the invention is given in Table 2 (SEQ ID NO:8).

The term “biosimilar” means a biological product, including a monoclonalantibody or fusion protein, that is highly similar to a U.S. licensedreference biological product notwithstanding minor differences inclinically inactive components, and for which there are no clinicallymeaningful differences between the biological product and the referenceproduct in terms of the safety, purity, and potency of the product.Furthermore, a similar biological or “biosimilar” medicine is abiological medicine that is similar to another biological medicine thathas already been authorized for use by the European Medicines Agency.The term “biosimilar” is also used synonymously by other national andregional regulatory agencies. Biological products or biologicalmedicines are medicines that are made by or derived from a biologicalsource, such as a bacterium or yeast. They can consist of relativelysmall molecules such as human insulin or erythropoietin, or complexmolecules such as monoclonal antibodies. For example, if the referenceIL-2 protein is aldesleukin (PROLEUKIN), a protein approved by drugregulatory authorities with reference to aldesleukin is a “biosimilarto” aldesleukin or is a “biosimilar thereof” of aldesleukin. In Europe,a similar biological or “biosimilar” medicine is a biological medicinethat is similar to another biological medicine that has already beenauthorized for use by the European Medicines Agency (EMA). The relevantlegal basis for similar biological applications in Europe is Article 6of Regulation (EC) No 726/2004 and Article 10(4) of Directive2001/83/EC, as amended and therefore in Europe, the biosimilar may beauthorized, approved for authorization or subject of an application forauthorization under Article 6 of Regulation (EC) No 726/2004 and Article10(4) of Directive 2001/83/EC. The already authorized originalbiological medicinal product may be referred to as a “referencemedicinal product” in Europe. Some of the requirements for a product tobe considered a biosimilar are outlined in the CHMP Guideline on SimilarBiological Medicinal Products. In addition, product specific guidelines,including guidelines relating to monoclonal antibody biosimilars, areprovided on a product-by-product basis by the EMA and published on itswebsite. A biosimilar as described herein may be similar to thereference medicinal product by way of quality characteristics,biological activity, mechanism of action, safety profiles and/orefficacy. In addition, the biosimilar may be used or be intended for useto treat the same conditions as the reference medicinal product. Thus, abiosimilar as described herein may be deemed to have similar or highlysimilar quality characteristics to a reference medicinal product.Alternatively, or in addition, a biosimilar as described herein may bedeemed to have similar or highly similar biological activity to areference medicinal product. Alternatively, or in addition, a biosimilaras described herein may be deemed to have a similar or highly similarsafety profile to a reference medicinal product. Alternatively, or inaddition, a biosimilar as described herein may be deemed to have similaror highly similar efficacy to a reference medicinal product. Asdescribed herein, a biosimilar in Europe is compared to a referencemedicinal product which has been authorized by the EMA. However, in someinstances, the biosimilar may be compared to a biological medicinalproduct which has been authorized outside the European Economic Area (anon-EEA authorized “comparator”) in certain studies. Such studiesinclude for example certain clinical and in vivo non-clinical studies.As used herein, the term “biosimilar” also relates to a biologicalmedicinal product which has been or may be compared to a non-EEAauthorized comparator. Certain biosimilars are proteins such asantibodies, antibody fragments (for example, antigen binding portions)and fusion proteins. A protein biosimilar may have an amino acidsequence that has minor modifications in the amino acid structure(including for example deletions, additions, and/or substitutions ofamino acids) which do not significantly affect the function of thepolypeptide. The biosimilar may comprise an amino acid sequence having asequence identity of 97% or greater to the amino acid sequence of itsreference medicinal product, e.g., 97%, 98%, 99% or 100%. The biosimilarmay comprise one or more post-translational modifications, for example,although not limited to, glycosylation, oxidation, deamidation, and/ortruncation which is/are different to the post-translationalmodifications of the reference medicinal product, provided that thedifferences do not result in a change in safety and/or efficacy of themedicinal product. The biosimilar may have an identical or differentglycosylation pattern to the reference medicinal product. Particularly,although not exclusively, the biosimilar may have a differentglycosylation pattern if the differences address or are intended toaddress safety concerns associated with the reference medicinal product.Additionally, the biosimilar may deviate from the reference medicinalproduct in for example its strength, pharmaceutical form, formulation,excipients and/or presentation, providing safety and efficacy of themedicinal product is not compromised. The biosimilar may comprisedifferences in for example pharmacokinetic (PK) and/or pharmacodynamic(PD) profiles as compared to the reference medicinal product but isstill deemed sufficiently similar to the reference medicinal product asto be authorized or considered suitable for authorization. In certaincircumstances, the biosimilar exhibits different binding characteristicsas compared to the reference medicinal product, wherein the differentbinding characteristics are considered by a Regulatory Authority such asthe EMA not to be a barrier for authorization as a similar biologicalproduct. The term “biosimilar” is also used synonymously by othernational and regional regulatory agencies.

As used herein, the term “variant” encompasses but is not limited toantibodies or fusion proteins which comprise an amino acid sequencewhich differs from the amino acid sequence of a reference antibody byway of one or more substitutions, deletions and/or additions at certainpositions within or adjacent to the amino acid sequence of the referenceantibody. The variant may comprise one or more conservativesubstitutions in its amino acid sequence as compared to the amino acidsequence of a reference antibody. Conservative substitutions mayinvolve, e.g., the substitution of similarly charged or uncharged aminoacids. The variant retains the ability to specifically bind to theantigen of the reference antibody. The term variant also includespegylated antibodies or proteins.

“Pegylation” refers to a modified antibody, or a fragment thereof, thattypically is reacted with polyethylene glycol (PEG), such as a reactiveester or aldehyde derivative of PEG, under conditions in which one ormore PEG groups become attached to the antibody, antibody fragment, orprotein. Pegylation may, for example, increase the biological (e.g.,serum) half life of the antibody or protein. Preferably, the pegylationis carried out via an acylation reaction or an alkylation reaction witha reactive PEG molecule (or an analogous reactive water-solublepolymer). As used herein, the term “polyethylene glycol” is intended toencompass any of the forms of PEG that have been used to derivatizeother proteins, such as mono (C₁-C₁₀) alkoxy- or aryloxy-polyethyleneglycol or polyethylene glycol-maleimide. The antibody or protein to bepegylated may be an aglycosylated antibody. Methods for pegylation areknown in the art and can be applied to the antibodies of the invention,as described for example in European Patent Nos. EP 0154316 and EP0401384.

The term “hematological malignancy” refers to mammalian cancers andtumors of the hematopoietic and lymphoid tissues, including but notlimited to tissues of the blood, bone marrow, lymph nodes, and lymphaticsystem. Hematological malignancies are also referred to as “liquidtumors.” Hematological malignancies include, but are not limited to,acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL),small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML),chronic myelogenous leukemia (CML), acute monocytic leukemia (AMoL),Hodgkin's lymphoma, and non-Hodgkin's lymphomas. The term “B cellhematological malignancy” refers to hematological malignancies thataffect B cells.

The term “solid tumor” refers to an abnormal mass of tissue that usuallydoes not contain cysts or liquid areas. Solid tumors may be benign ormalignant. The term “solid tumor cancer” refers to malignant,neoplastic, or cancerous solid tumors. Solid tumor cancers include, butare not limited to, sarcomas, carcinomas, and lymphomas, such as cancersof the lung, breast, prostate, colon, rectum, and bladder. The tissuestructure of solid tumors includes interdependent tissue compartmentsincluding the parenchyma (cancer cells) and the supporting stromal cellsin which the cancer cells are dispersed and which may provide asupporting microenvironment.

The term “liquid tumor” refers to an abnormal mass of cells that isfluid in nature. Liquid tumor cancers include, but are not limited to,leukemias, myelomas, and lymphomas, as well as other hematologicalmalignancies. TILs obtained from liquid tumors may also be referred toherein as marrow infiltrating lymphocytes (MILs).

The term “microenvironment,” as used herein, may refer to the solid orhematological tumor microenvironment as a whole or to an individualsubset of cells within the microenvironment. The tumor microenvironment,as used herein, refers to a complex mixture of “cells, soluble factors,signaling molecules, extracellular matrices, and mechanical cues thatpromote neoplastic transformation, support tumor growth and invasion,protect the tumor from host immunity, foster therapeutic resistance, andprovide niches for dominant metastases to thrive,” as described inSwartz, et al., Cancer Res., 2012, 72, 2473. Although tumors expressantigens that should be recognized by T cells, tumor clearance by theimmune system is rare because of immune suppression by themicroenvironment.

The terms “fragmenting,” “fragment,” and “fragmented,” as used herein todescribe processes for disrupting a tumor, includes mechanicalfragmentation methods such as crushing, slicing, dividing, andmorcellating tumor tissue as well as any other method for disrupting thephysical structure of tumor tissue.

The terms “about” and “approximately” mean within a statisticallymeaningful range of a value. Such a range can be within an order ofmagnitude, preferably within 50%, more preferably within 20%, morepreferably still within 10%, and even more preferably within 5% of agiven value or range. The allowable variation encompassed by the terms“about” or “approximately” depends on the particular system under study,and can be readily appreciated by one of ordinary skill in the art.Moreover, as used herein, the terms “about” and “approximately” meanthat dimensions, sizes, formulations, parameters, shapes and otherquantities and characteristics are not and need not be exact, but may beapproximate and/or larger or smaller, as desired, reflecting tolerances,conversion factors, rounding off, measurement error and the like, andother factors known to those of skill in the art. In general, adimension, size, formulation, parameter, shape or other quantity orcharacteristic is “about” or “approximate” whether or not expresslystated to be such. It is noted that embodiments of very different sizes,shapes and dimensions may employ the described arrangements.

The transitional terms “comprising,” “consisting essentially of,” and“consisting of,” when used in the appended claims, in original andamended form, define the claim scope with respect to what unrecitedadditional claim elements or steps, if any, are excluded from the scopeof the claim(s). The term “comprising” is intended to be inclusive oropen-ended and does not exclude any additional, unrecited element,method, step or material. The term “consisting of” excludes any element,step or material other than those specified in the claim and, in thelatter instance, impurities ordinary associated with the specifiedmaterial(s). The term “consisting essentially of” limits the scope of aclaim to the specified elements, steps or material(s) and those that donot materially affect the basic and novel characteristic(s) of theclaimed invention. All compositions, methods, and kits described hereinthat embody the invention can, in alternate embodiments, be morespecifically defined by any of the transitional terms “comprising,”“consisting essentially of,” and “consisting of.”

For the avoidance of doubt, it is intended herein that particularfeatures (for example integers, characteristics, values, uses, diseases,formulae, compounds or groups) described in conjunction with aparticular aspect, embodiment or example of the invention are to beunderstood as applicable to any other aspect, embodiment or exampledescribed herein unless incompatible therewith. Thus such features maybe used where appropriate in conjunction with any of the definition,claims or embodiments defined herein. All of the features disclosed inthis specification (including any accompanying claims, abstract anddrawings), and/or all of the steps of any method or process sodisclosed, may be combined in any combination, except combinations whereat least some of the features and/or steps are mutually exclusive. Theinvention is not restricted to any details of any disclosed embodiments.The invention extends to any novel one, or novel combination, of thefeatures disclosed in this specification (including any accompanyingclaims, abstract and drawings), or to any novel one, or any novelcombination, of the steps of any method or process so disclosed.

Methods of Expanding Remnant Tumor Infiltrating Lymphocytes

In an embodiment, the invention includes a method of expanding remnanttumor infiltrating lymphocytes (rTILs) after digestion of a tumor asdescribed herein.

In an embodiment, the invention includes a method of expanding rTILs,the method comprising contacting a population of rTILs comprising atleast one rTIL with IL-2, thereby expanding rTILs.

In an embodiment, the invention provides a method of expanding apopulation of rTILs, the method comprising the steps as described inJin, et al., J. Immunotherapy 2012, 35, 283-292, the disclosure of whichis incorporated by reference herein. For example, the tumor may beplaced in enzyme media and mechanically fragmented for approximately 1minute. The mixture may then be incubated for 30 minutes at 37° C. in 5%CO₂ and then mechanically fragmented again for approximately 1 minute.After incubation for 30 minutes at 37° C. in 5% CO₂, the tumor may bemechanically fragmented a third time for approximately 1 minute. Ifafter the third mechanical disruption, large pieces of tissue arepresent, 1 or 2 additional mechanical dissociations may be applied tothe sample, with or without 30 additional minutes of incubation at 37°C. in 5% CO₂. At the end of the final incubation, if the cell suspensioncontains a large number of red blood cells or dead cells, a densitygradient separation using Ficoll may be performed to remove these cells.TIL cultures were initiated in 24-well plates (Costar 24-well cellculture cluster, flat bottom; Corning Incorporated, Corning, N.Y.), eachwell may be seeded with 1×10⁶ tumor digest cells or one tumor fragmentapproximately 1-8 mm³ in size in 2 mL of complete medium (CM) with IL-2(6000 IU/mL; Chiron Corp., Emeryville, Calif.). CM consists of RPMI 1640with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10mg/mL gentamicin. Cultures may be initiated in gas-permeable flasks witha 40 mL capacity and a 10 cm² gas-permeable silicon bottom (G-Rex 10;Wilson Wolf Manufacturing, New Brighton), each flask may be loaded with10-40×10⁶ viable tumor digest cells or 5-30 tumor fragments in 10-40 mLof CM with IL-2. G-Rex 10 and 24-well plates may be incubated in ahumidified incubator at 37° C. in 5% CO₂ and 5 days after cultureinitiation, half the media may be removed and replaced with fresh CM andIL-2 and after day 5, half the media may be changed every 2-3 days. Arapid expansion protocol (REP) for TILs may be performed using T-175flasks and gas-permeable bags or gas-permeable G-Rex flasks, asdescribed elsewhere herein. For REP in T-175 flasks, 1×10⁶ rTILs may besuspended in 150 mL of media in each flask. The rTIL may be cultured ina 1 to 1 mixture of CM and AIM-V medium (50/50 medium), supplementedwith 3000 IU/mL of IL-2 and 30 ng/mL of anti-CD3 antibody (OKT-3). TheT-175 flasks may be incubated at 37° C. in 5% CO₂. Half the media may bechanged on day 5 using 50/50 medium with 3000 IU/mL of IL-2. On day 7,cells from 2 T-175 flasks may be combined in a 3 L bag and 300 mL ofAIM-V with 5% human AB serum and 3000 IU/mL of IL-2 may be added to the300 mL of TIL suspension. The number of cells in each bag may be countedevery day or two days, and fresh media may be added to keep the cellcount between 0.5 and 2.0×10⁶ cells/mL. For REP in 500 mL capacityflasks with 100 cm² gas-permeable silicon bottoms (e.g., G-Rex 100,Wilson Wolf Manufacturing, as described elsewhere herein), 5×10⁶ or10×10⁶ TILs may be cultured in 400 mL of 50/50 medium, supplemented with3000 IU/mL of IL-2 and 30 ng/mL of anti-CD3 antibody (OKT-3). TheG-Rex100 flasks may be incubated at 37° C. in 5% CO₂. On day five, 250mL of supernatant may be removed and placed into centrifuge bottles andcentrifuged at 1500 rpm (491 g) for 10 minutes. The obtained TIL pelletsmay be resuspended with 150 mL of fresh 50/50 medium with 3000 IU/mL ofIL-2 and added back to the G-Rex 100 flasks. When TIL are expandedserially in G-Rex 100 flasks, on day seven the TIL in each G-Rex100 aresuspended in the 300 mL of media present in each flask and the cellsuspension may be divided into three 100 mL aliquots that may be used toseed 3 G-Rex100 flasks. About 150 mL of AIM-V with 5% human AB serum and3000 IU/mL of IL-2 may then be added to each flask. G-Rex100 flasks maythen be incubated at 37° C. in 5% CO₂, and after four days, 150 mL ofAIM-V with 3000 IU/mL of IL-2 may be added to each G-Rex100 flask. Afterthis, the REP may be completed by harvesting cells on day 14 of culture.

In an embodiment, a method of expanding or treating a cancer includes astep wherein TILs are obtained from a patient tumor sample. A patienttumor sample may be obtained using methods known in the art. Forexample, TILs may be cultured from enzymatic tumor digests and tumorfragments (about 1 to about 8 mm³ in size) from sharp dissection. Suchtumor digests may be produced by incubation in enzymatic media (e.g.,Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10mcg/mL gentamicine, 30 units/mL of DNase and 1.0 mg/mL of collagenase)followed by mechanical dissociation (e.g., using a tissue dissociator orfragmenter). Tumor digests may be produced by placing the tumor inenzymatic media and mechanically fragmenting the tumor for approximately1 minute, followed by incubation for 30 minutes at 37° C. in 5% CO₂,followed by repeated cycles of mechanical dissociation and incubationunder the foregoing conditions until only small tissue pieces arepresent. At the end of this process, if the cell suspension contains alarge number of red blood cells or dead cells, a density gradientseparation using FICOLL branched hydrophilic polysaccharide may beperformed to remove these cells. Alternative methods known in the artmay be used, such as those described in U.S. Patent ApplicationPublication No. 2012/0244133 A1, the disclosure of which is incorporatedby reference herein. Any of the foregoing methods may be used in any ofthe embodiments described herein for methods of expanding TILs ormethods treating a cancer.

In an embodiment, REP of rTILs can be performed in a gas permeablecontainer using any suitable method. For example, rTILs can be rapidlyexpanded using non-specific T cell receptor stimulation in the presenceof interleukin-2 (IL-2), interleukin-15 (IL-15), and/or interleukin-21(IL-21), as described, e.g., in International Patent ApplicationPublication Nos. WO 2015/189356 A1 and WO 2015/189356 A1, thedisclosures of each of which are incorporated by reference herein. Thenon-specific T cell receptor stimulus can include, for example, about 30ng/mL of OKT-3, a monoclonal anti-CD3 antibody (commercially availablefrom Ortho-McNeil, Raritan, N.J. or Miltenyi Biotech, Inc., San Diego,Calif., USA). TILs can be rapidly expanded by further stimulation of theTILs in vitro with one or more antigens, including antigenic portionsthereof, such as epitope(s), of the cancer, which can be optionallyexpressed from a vector, such as a human leukocyte antigen A2 (HLA-A2)binding peptide, e.g., 0.3 μM MART-1:26-35 (27 L) or gpl 00:209-217(210M), optionally in the presence of a T cell growth factor, such as300 IU/mL IL-2 or IL-15. Other suitable antigens may include, e.g.,NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, andVEGFR2, or antigenic portions thereof. TIL may also be rapidly expandedby re-stimulation with the same antigen(s) of the cancer pulsed ontoHLA-A2-expressing antigen-presenting cells. Alternatively, the TILs canbe further re-stimulated with, e.g., example, irradiated, autologouslymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

In an embodiment, a method for expanding TILs may include using about5000 mL to about 25000 mL of cell medium, about 5000 mL to about 10000mL of cell culture medium, or about 5800 mL to about 8700 mL of cellculture medium. In an embodiment, a method for expanding TILs mayinclude using about 1000 mL to about 2000 mL of cell medium, about 2000mL to about 3000 mL of cell culture medium, about 3000 mL to about 4000mL of cell culture medium, about 4000 mL to about 5000 mL of cellculture medium, about 5000 mL to about 6000 mL of cell culture medium,about 6000 mL to about 7000 mL of cell culture medium, about 7000 mL toabout 8000 mL of cell culture medium, about 8000 mL to about 9000 mL ofcell culture medium, about 9000 mL to about 10000 mL of cell culturemedium, about 10000 mL to about 15000 mL of cell culture medium, about15000 mL to about 20000 mL of cell culture medium, or about 20000 mL toabout 25000 mL of cell culture medium. In an embodiment, expanding thenumber of TILs uses no more than one type of cell culture medium. Anysuitable cell culture medium may be used, e.g., AIM-V cell medium(L-glutamine, 50 μM streptomycin sulfate, and 10 μM gentamicin sulfate)cell culture medium (Invitrogen, Carlsbad Calif.). In this regard, theinventive methods advantageously reduce the amount of medium and thenumber of types of medium required to expand the number of TIL. In anembodiment, expanding the number of TIL may comprise feeding the cellsno more frequently than every third or fourth day. Expanding the numberof cells in a gas permeable container simplifies the proceduresnecessary to expand the number of cells by reducing the feedingfrequency necessary to expand the cells.

In an embodiment, the rapid expansion is performed using a gas permeablecontainer. Such embodiments allow for cell populations to expand fromabout 5×10⁵ cells/cm² to between 10×10⁶ and 30×10⁶ cells/cm². In anembodiment, this expansion occurs without feeding. In an embodiment,this expansion occurs without feeding so long as medium resides at aheight of about 10 cm in a gas-permeable flask. In an embodiment this iswithout feeding but with the addition of one or more cytokines. In anembodiment, the cytokine can be added as a bolus without any need to mixthe cytokine with the medium. Such containers, devices, and methods areknown in the art and have been used to expand TILs, and include thosedescribed in U.S. Patent Application Publication No. US 2014/0377739 A1,International Patent Application Publication No. WO 2014/210036 A1, U.S.Patent Application Publication No. US 2013/0115617 A1, InternationalPublication No. WO 2013/188427 A1, U.S. Patent Application PublicationNo. US 2011/0136228 A1, U.S. Pat. No. 8,809,050, International PatentApplication Publication No. WO 2011/072088 A2, U.S. Patent ApplicationPublication No. US 2016/0208216 A1, U.S. Patent Application PublicationNo. US 2012/0244133 A1, International Patent Application Publication No.WO 2012/129201 A1, U.S. Patent Application Publication No. US2013/0102075 A1, U.S. Pat. No. 8,956,860, International PatentApplication Publication No. WO 2013/173835 A1, and U.S. PatentApplication Publication No. US 2015/0175966 A1, the disclosures of whichare incorporated herein by reference. Such processes are also describedin Jin, et al., J. Immunotherapy 2012, 35, 283-292, the disclosure ofwhich is incorporated by reference herein.

In an embodiment, the gas permeable container is a G-Rex 10 flask(Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In anembodiment, the gas permeable container includes a 10 cm² gas permeableculture surface. In an embodiment, the gas permeable container includesa 40 mL cell culture medium capacity. In an embodiment, the gaspermeable container provides 100 to 300 million TILs after 2 mediumexchanges.

In an embodiment, the gas permeable container is a G-Rex 100 flask(Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In anembodiment, the gas permeable container includes a 100 cm² gas permeableculture surface. In an embodiment, the gas permeable container includesa 450 mL cell culture medium capacity. In an embodiment, the gaspermeable container provides 1 to 3 billion TILs after 2 mediumexchanges.

In an embodiment, the gas permeable container is a G-Rex 100M flask(Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In anembodiment, the gas permeable container includes a 100 cm² gas permeableculture surface. In an embodiment, the gas permeable container includesa 1000 mL cell culture medium capacity. In an embodiment, the gaspermeable container provides 1 to 3 billion TILs without mediumexchange.

In an embodiment, the gas permeable container is a G-Rex 100 L flask(Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In anembodiment, the gas permeable container includes a 100 cm² gas permeableculture surface. In an embodiment, the gas permeable container includesa 2000 mL cell culture medium capacity. In an embodiment, the gaspermeable container provides 1 to 3 billion TILs without mediumexchange.

In an embodiment, the gas permeable container is a G-Rex 24 well plate(Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In anembodiment, the gas permeable container includes a plate with wells,wherein each well includes a 2 cm² gas permeable culture surface. In anembodiment, the gas permeable container includes a plate with wells,wherein each well includes a 8 mL cell culture medium capacity. In anembodiment, the gas permeable container provides 20 to 60 million cellsper well after 2 medium exchanges.

In an embodiment, the gas permeable container is a G-Rex 6 well plate(Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In anembodiment, the gas permeable container includes a plate with wells,wherein each well includes a 10 cm² gas permeable culture surface. In anembodiment, the gas permeable container includes a plate with wells,wherein each well includes a 40 mL cell culture medium capacity. In anembodiment, the gas permeable container provides 100 to 300 millioncells per well after 2 medium exchanges.

In an embodiment, the cell medium in the first and/or second gaspermeable container is unfiltered. The use of unfiltered cell medium maysimplify the procedures necessary to expand the number of cells. In anembodiment, the cell medium in the first and/or second gas permeablecontainer lacks beta-mercaptoethanol (BME).

In an embodiment, the duration of the method comprising obtaining atumor tissue sample from the mammal; culturing the tumor tissue samplein a first gas permeable container containing cell medium therein;obtaining TILs from the tumor tissue sample; expanding the number ofTILs in a second gas permeable container containing cell medium thereinfor a duration of about 14 to about 42 days, e.g., about 28 days.

In an embodiment, the ratio of rTILs to PBMCs in the rapid expansion isabout 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250,about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1to 375, about 1 to 400, or about 1 to 500. In an embodiment, the ratioof rTILs to PBMCs in the rapid expansion is between 1 to 50 and 1 to300. In an embodiment, the ratio of rTILs to PBMCs in the rapidexpansion is between 1 to 100 and 1 to 200.

In an embodiment, the ratio of rTILs to PBMCs (rTIL:PBMC) is selectedfrom the group consisting of 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35,1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95,1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145,1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195,1:200, 1:225, 1:250, 1:275, 1:300, 1:350, 1:400, 1:450, and 1:500. In apreferred embodiment, the ratio of rTILs to PBMCs (rTIL:PBMC) is about1:90. In a preferred embodiment, the ratio of TILs to PBMCs (rTIL:PBMC)is about 1:95. In a preferred embodiment, the ratio of rTILs to PBMCs(TIL:PBMC) is about 1:100. In a preferred embodiment, the ratio of rTILsto PBMCs (TIL:PBMC) is about 1:105. In a preferred embodiment, the ratioof rTILs to PBMCs (TIL:PBMC) is about 1:110.

In an embodiment, the cell culture medium further comprises IL-2. In apreferred embodiment, the cell culture medium comprises about 3000 IU/mLof IL-2. In an embodiment, the cell culture medium comprises about 1000IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000IU/mL, about 7500 IU/mL, or about 8000 IU/mL of IL-2. In an embodiment,the cell culture medium comprises between 1000 and 2000 IU/mL, between2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between7000 and 8000 IU/mL, or between 8000 IU/mL of IL-2.

In an embodiment, the cell culture medium comprises OKT-3 antibody. In apreferred embodiment, the cell culture medium comprises about 30 ng/mLof OKT-3 antibody. In an embodiment, the cell culture medium comprisesabout 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL,about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1μg/mL of OKT-3 antibody. In an embodiment, the cell culture mediumcomprises between 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL,between 5 ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20ng/mL and 30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40 ng/mL and50 ng/mL, and between 50 ng/mL and 100 ng/mL of OKT-3 antibody.

In an embodiment, a rapid expansion process for TILs may be performedusing T-175 flasks and gas permeable bags as previously described (Tran,et al., J. Immunother. 2008, 31, 742-51; Dudley, et al., J. Immunother.2003, 26, 332-42) or gas permeable cultureware (G-Rex flasks,commercially available from Wilson Wolf Manufacturing Corporation, NewBrighton, Minn., USA). For TIL rapid expansion in T-175 flasks, 1×10⁶TILs suspended in 150 mL of media may be added to each T-175 flask. TheTILs may be cultured in a 1 to 1 mixture of CM and AIM-V medium,supplemented with 3000 IU (injection units) per mL of IL-2 and 30 ng permL of anti-CD3 antibody (e.g., OKT-3). The T-175 flasks may be incubatedat 37° C. in 5% CO₂. Half the media may be exchanged on day 5 using50/50 medium with 3000 IU per mL of IL-2. On day 7 cells from two T-175flasks may be combined in a 3 liter bag and 300 mL of AIM V with 5%human AB serum and 3000 IU per mL of IL-2 was added to the 300 ml of TILsuspension. The number of cells in each bag was counted every day or twoand fresh media was added to keep the cell count between 0.5 and 2.0×10⁶cells/mL.

In an embodiment, for TIL rapid expansions in 500 mL capacity gaspermeable flasks with 100 cm gas-permeable silicon bottoms (G-Rex 100,commercially available from Wilson Wolf Manufacturing Corporation, NewBrighton, Minn., USA), 5×10⁶ or 10×10⁶ TIL may be cultured in 50/50medium, supplemented with 5% human AB serum, 3000 IU per mL of IL-2 and30 ng per mL of anti-CD3 (OKT-3). The G-Rex 100 flasks may be incubatedat 37° C. in 5% CO₂. On day 5, 250 mL of supernatant may be removed andplaced into centrifuge bottles and centrifuged at 1500 rpm (revolutionsper minute; 491×g) for 10 minutes. The TIL pellets may be re-suspendedwith 150 mL of fresh medium with 5% human AB serum, 3000 IU per mL ofIL-2, and added back to the original G-Rex 100 flasks. When TIL areexpanded serially in G-Rex 100 flasks, on day 7 the TIL in each G-Rex100 may be suspended in the 300 mL of media present in each flask andthe cell suspension may be divided into 3 100 mL aliquots that may beused to seed 3 G-Rex 100 flasks. Then 150 mL of AIM-V with 5% human ABserum and 3000 IU per mL of IL-2 may be added to each flask. The G-Rex100 flasks may be incubated at 37° C. in 5% CO₂ and after 4 days 150 mLof AIM-V with 3000 IU per mL of IL-2 may be added to each G-Rex 100flask. The cells may be harvested on day 14 of culture.

In an embodiment, TILs may be prepared as follows. 2 mm³ tumor fragmentsare cultured in complete media (CM) comprised of AIM-V medium(Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 2 mMglutamine (Mediatech, Inc. Manassas, Va.), 100 U/mL penicillin(Invitrogen Life Technologies), 100 μg/mL streptomycin (Invitrogen LifeTechnologies), 5% heat-inactivated human AB serum (Valley Biomedical,Inc. Winchester, Va.) and 600 IU/mL rhIL-2 (Chiron, Emeryville, Calif.).For enzymatic digestion of solid tumors, tumor specimens was diced intoRPMI-1640, washed and centrifuged at 800 rpm for 5 minutes at 15-22° C.,and resuspended in enzymatic digestion buffer (0.2 mg/mL Collagenase and30 units/ml of DNase in RPMI-1640) followed by overnight rotation atroom temperature. TILs established from fragments may be grown for 3-4weeks in CM and expanded fresh or cryopreserved in heat-inactivated HABserum with 10% dimethylsulfoxide (DMSO) and stored at −180° C. until thetime of study. Tumor associated lymphocytes (TAL) obtained from ascitescollections were seeded at 3×10⁶ cells/well of a 24 well plate in CM.TIL growth was inspected about every other day using a low-powerinverted microscope.

In an embodiment, TILs are expanded in gas-permeable containers.Gas-permeable containers have been used to expand TILs using PBMCs usingmethods, compositions, and devices known in the art, including thosedescribed in U.S. Patent Application Publication No. U.S. PatentApplication Publication No. 2005/0106717 A1, the disclosures of whichare incorporated herein by reference. In an embodiment, TILs areexpanded in gas-permeable bags. In an embodiment, TILs are expandedusing a cell expansion system that expands TILs in gas permeable bags,such as the Xuri Cell Expansion System W25 (GE Healthcare). In anembodiment, TILs are expanded using a cell expansion system that expandsTILs in gas permeable bags, such as the WAVE Bioreactor System, alsoknown as the Xuri Cell Expansion System W5 (GE Healthcare). In anembodiment, the cell expansion system includes a gas permeable cell bagwith a volume selected from the group consisting of about 100 mL, about200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L,about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about10 L, about 11 L, about 12 L, about 13 L, about 14 L, about 15 L, about16 L, about 17 L, about 18 L, about 19 L, about 20 L, about 25 L, andabout 30 L. In an embodiment, the cell expansion system includes a gaspermeable cell bag with a volume range selected from the groupconsisting of between 50 and 150 mL, between 150 and 250 mL, between 250and 350 mL, between 350 and 450 mL, between 450 and 550 mL, between 550and 650 mL, between 650 and 750 mL, between 750 and 850 mL, between 850and 950 mL, and between 950 and 1050 mL. In an embodiment, the cellexpansion system includes a gas permeable cell bag with a volume rangeselected from the group consisting of between 1 L and 2 L, between 2 Land 3 L, between 3 L and 4 L, between 4 L and 5 L, between 5 L and 6 L,between 6 L and 7 L, between 7 L and 8 L, between 8 L and 9 L, between 9L and 10 L, between 10 L and 11 L, between 11 L and 12 L, between 12 Land 13 L, between 13 L and 14 L, between 14 L and 15 L, between 15 L and16 L, between 16 L and 17 L, between 17 L and 18 L, between 18 L and 19L, and between 19 L and 20 L. In an embodiment, the cell expansionsystem includes a gas permeable cell bag with a volume range selectedfrom the group consisting of between 0.5 L and 5 L, between 5 L and 10L, between 10 L and 15 L, between 15 L and 20 L, between 20 L and 25 L,and between 25 L and 30 L. In an embodiment, the cell expansion systemutilizes a rocking time of about 30 minutes, about 1 hour, about 2hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours,about 12 hours, about 24 hours, about 2 days, about 3 days, about 4days, about 5 days, about 6 days, about 7 days, about 8 days, about 9days, about 10 days, about 11 days, about 12 days, about 13 days, about14 days, about 15 days, about 16 days, about 17 days, about 18 days,about 19 days, about 20 days, about 21 days, about 22 days, about 23days, about 24 days, about 25 days, about 26 days, about 27 days, andabout 28 days. In an embodiment, the cell expansion system utilizes arocking time of between 30 minutes and 1 hour, between 1 hour and 12hours, between 12 hours and 1 day, between 1 day and 7 days, between 7days and 14 days, between 14 days and 21 days, and between 21 days and28 days. In an embodiment, the cell expansion system utilizes a rockingrate of about 2 rocks/minute, about 5 rocks/minute, about 10rocks/minute, about 20 rocks/minute, about 30 rocks/minute, and about 40rocks/minute. In an embodiment, the cell expansion system utilizes arocking rate of between 2 rocks/minute and 5 rocks/minute, 5rocks/minute and 10 rocks/minute, 10 rocks/minute and 20 rocks/minute,20 rocks/minute and 30 rocks/minute, and 30 rocks/minute and 40rocks/minute. In an embodiment, the cell expansion system utilizes arocking angle of about 2°, about 3°, about 4°, about 5°, about 6°, about7°, about 8°, about 9°, about 10°, about 11°, and about 12°. In anembodiment, the cell expansion system utilizes a rocking angle ofbetween 2° and 3°, between 3° and 4°, between 4° and 5°, between 5° and6°, between 6° and 7°, between 7° and 8°, between 8° and 9°, between 9°and 10°, between 10° and 11°, and between 11° and 12°.

In an embodiment, a method of expanding rTILs further comprises a stepwherein rTILs are selected for superior tumor reactivity. Any selectionmethod known in the art may be used. For example, the methods describedin U.S. Patent Application Publication No. 2016/0010058 A1, thedisclosures of which are incorporated herein by reference, may be usedfor selection of TILs for superior tumor reactivity.

Characteristics of rTILs

In an embodiment, the rTILs of the invention exhibit an exhausted T cellphenotype characterized by one or more T cell exhaustion markers. In anembodiment, the rTILs of the invention exhibit an exhausted T cellphenotype characterized by one or more T cell exhaustion markers usingflow cytometry analysis. In an embodiment, the T cell exhaustion markeris PD-1. In an embodiment, the T cell exhaustion marker is LAG3. In anembodiment, the T cell exhaustion marker is TIM3.

In an embodiment, PD-1 expression in rTILs is reduced by at least 5%, atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 100%, atleast 110%, at least 120%, at least 130%, at least 140%, or at least150% relative to the eTILs. In an embodiment, PD-1 expression in rTILsis reduced by at least 2-fold, at least 3-fold, at least 4-fold, atleast 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, atleast 9-fold, or at least 10-fold relative to the eTILs.

In an embodiment, LAG3 expression in rTILs is reduced by at least 5%, atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 100%, atleast 110%, at least 120%, at least 130%, at least 140%, or at least150% relative to the eTILs. In an embodiment, LAG3 expression in rTILsis reduced by at least 2-fold, at least 3-fold, at least 4-fold, atleast 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, atleast 9-fold, or at least 10-fold relative to the eTILs. In anembodiment, LAG3 expression in rTILs is undetectable by flow cytometry.

In an embodiment, TIM3 expression in rTILs is reduced by at least 5%, atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 100%, atleast 110%, at least 120%, at least 130%, at least 140%, or at least150% relative to the eTILs. In an embodiment, TIM3 expression in rTILsis reduced by at least 2-fold, at least 3-fold, at least 4-fold, atleast 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, atleast 9-fold, or at least 10-fold relative to the eTILs. In anembodiment, TIM3 expression in rTILs is undetectable by flow cytometry.

In an embodiment, TIGIT expression in rTILs is reduced by at least 5%,at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 100%, atleast 110%, at least 120%, at least 130%, at least 140%, or at least150% relative to the eTILs. In an embodiment, TIGIT expression in rTILsis reduced by at least 2-fold, at least 3-fold, at least 4-fold, atleast 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, atleast 9-fold, or at least 10-fold relative to the eTILs. In anembodiment, TIGIT expression in rTILs is undetectable by flow cytometry.

In an embodiment, CTLA-4 expression in rTILs is reduced by at least 5%,at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 100%, atleast 110%, at least 120%, at least 130%, at least 140%, or at least150% relative to the eTILs. In an embodiment, CTLA-4 expression in rTILsis reduced by at least 2-fold, at least 3-fold, at least 4-fold, atleast 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, atleast 9-fold, or at least 10-fold relative to the eTILs. In anembodiment, CTLA-4 expression in rTILs is undetectable by flowcytometry.

In an embodiment, CD69 expression in rTILs is increased by at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 100%, at least 110%, atleast 120%, at least 130%, at least 140%, or at least 150% relative tothe eTILs. In an embodiment, CD69 expression in rTILs is increased by atleast 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, atleast 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or atleast 10-fold relative to the eTILs.

In an embodiment, S1PR1 (sphingosine-1-phosphate receptor 1) expressionin rTILs is decreased by at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 100%, at least 110%, at least 120%, at least 130%,at least 140%, or at least 150% relative to the eTILs. In an embodiment,S1PR1 expression in rTILs is decreased by at least 2-fold, at least3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relativeto the eTILs.

In an embodiment, telomere length in rTILs is increased by at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 100%, at least 110%, atleast 120%, at least 130%, at least 140%, or at least 150% relative tothe eTILs. In an embodiment, telomere length in rTILs is increased by atleast 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, atleast 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or atleast 10-fold relative to the eTILs.

In an embodiment, CD28 expression in rTILs is increased by at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 100%, at least 110%, atleast 120%, at least 130%, at least 140%, or at least 150% relative tothe eTILs. In an embodiment, CD28 expression in rTILs is increased by atleast 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, atleast 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or atleast 10-fold relative to the eTILs.

In an embodiment, CD27 expression in rTILs is increased by at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 100%, at least 110%, atleast 120%, at least 130%, at least 140%, or at least 150% relative tothe eTILs. In an embodiment, CD27 expression in rTILs is increased by atleast 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, atleast 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or atleast 10-fold relative to the eTILs.

In some embodiments, the methods described herein may include anoptional cryopreservation of eTIL and/or rTIL in a storage media (forexample, media containing 5% DMSO) prior to performing an additionalstep desribed herein or after completion of a REP step described herein,prior to transport, thawing, and/or administration to a patient. In someembodiments, the methods described herein may include a step of thawingcryopreserved TILs (e.g. cryopreserved eTIL, cryopreserved rTIL, or acombination or mixture thereof) prior to performing an additional stepdescribed herein. In some embodiments, the additional step may be anadditional or repeated expansion of the eTIL and/or rTIL (e.g., areREP), which may be performed on the thawed cells, using, for example,a supplemented cell culture medium comprising IL-2, OKT-3, and/or feedercells (e.g., antigen presenting cells), generally comprising peripheralblood mononuclear cells (PBMCs; or, alternatively, using antigenpresenting cells), wherein the additional expansion step may beperformed for at least 14 days. In some embodiments, such media may alsocontain combinations of IL-2, IL-15, and/or IL-23 rather than IL-2alone.

As discussed herein, cryopreservation can occur at numerous pointsthroughout the TIL expansion process. In some embodiments, a bulk TILpopulation (e.g., eTILs, rTILs, or a combination or mixture thereof)after expansion can be cryopreserved. Cryopreservation can be generallyaccomplished by placing the TIL population into a freezing solution,e.g., 85% complement inactivated AB serum and 15% dimethyl sulfoxide(DMSO). The cells in solution are placed into cryogenic vials and storedfor 24 hours at −80° C., with optional transfer to gaseous nitrogenfreezers for cryopreservation. See, Sadeghi, et al., Acta Oncologica2013, 52, 978-986. In some embodiments, the TILs described herein may becryopreserved in 5% DMSO. In some embodiments, the TILs described hereinmay be cryopreserved in cell culture media plus 5% DMSO.

When appropriate, the cryopreserved cells described herein, such ascryopreserved rTILs, are removed from the freezer and thawed in a 37° C.water bath until approximately ⅘ of the solution is thawed. The cellsare generally resuspended in complete media and optionally washed one ormore times. In some embodiments, the thawed TILs can be counted andassessed for viability as is known in the art.

Methods of Digesting Tumors to Obtain rTILs

In an embodiment, a method of obtaining rTILs includes a step wherein atumor is digested using one or more enzymes. Enzymes suitable fordigestion of tumors are described in Volvitz, et al., BMC Neuroscience2016, 17, 30, the disclosure of which is incorporated by referenceherein.

In some embodiments, the invention may include methods of obtainingrTILs that include a step wherein a tumor, which may include tumortissue or a portion thereof, is digested using an deoxyribonuclease, acollagenase, a hyaluronidase, or a combination thereof.

In an embodiment, a method of obtaining rTILs includes a step wherein atumor is digested using any enzyme that catalyzes the hydrolyticcleavage of phosphodiester linkages in the DNA backbone, thus degradingDNA. In an embodiment, a method of obtaining rTILs includes a stepwherein a tumor is digested using a deoxyribonuclease (DNase). In anembodiment, a method of obtaining rTILs includes a step wherein a tumoris digested using a deoxyribonuclease and at least one other enzyme. Inan embodiment, the deoxyribonuclease is deoxyribonuclease I. In anembodiment, the deoxyribonuclease is deoxyribonuclease II. In anembodiment, the deoxyribonuclease is deoxyribonuclease I from bovinepancreas (Sigma D5025 or equivalent). In an embodiment, thedeoxyribonuclease is recombinant deoxyribonuclease I from bovineexpressed in Pichia pastoris (Sigma D2821 or equivalent). In anembodiment, the deoxyribonuclease is recombinant human deoxyribonucleaseI (rhDNAase I, also known as dornase alfa, commercially available asPULMOZYME from Genentech, Inc.). In an embodiment, the deoxyribonucleaseis deoxyribonuclease II from bovine spleen (Sigma D8764 or equivalent).In an embodiment, the deoxyribonuclease is deoxyribonuclease II fromporcine spleen (Sigma D4138 or equivalent). In an embodiment, any of theforegoing deoxyribonucleases is present in the tumor digest. Thepreparation and properties of deoxyribonucleases suitable for use in theinvention are described in U.S. Pat. Nos. 5,783,433; 6,391,607;7,407,785; and 7,297,526, and International Patent ApplicationPublication No. WO 2016/108244 A1, the disclosures of each of which areincorporated by reference herein.

In an embodiment, a method of obtaining rTILs includes a step wherein atumor is digested using any enzyme that catalyzes the cleavage ofpeptide linkages in collagen, thus degrading collagen. In an embodiment,a method of obtaining rTILs includes a step wherein a tumor is digestedusing a collagenase. In an embodiment, a method of obtaining rTILsincludes a step wherein a tumor is digested using a collagenase and atleast one other enzyme. In an embodiment, the collagenase is collagenasefrom Clostridium histolyticum. In an embodiment, the collagenase isClostridiopeptidase A. In an embodiment, the collagenase is collagenaseI. In an embodiment, the collagenase is collagenase II. In anembodiment, the collagenase is collagenase from Clostridium histolyticum(Sigma C5138 or equivalent). The preparation and properties ofcollagenases suitable for use in the invention are described in U.S.Pat. Nos. 3,201,325; 3,705,083; 3,821,364; 5,177,017; 5,422,261;5,989,888; 9,211,316; the disclosures of each of which are incorporatedby reference herein.

In an embodiment, a method of obtaining rTILs includes a step wherein atumor is digested using any enzyme that catalyzes the degradation ofhyaluronic acid. In an embodiment, a method of obtaining rTILs includesa step wherein a tumor is digested using a hyaluronidase. In anembodiment, a method of obtaining rTILs includes a step wherein a tumoris digested using a hyaluronoglucosidase. In an embodiment, thehyaluronidase is hyaluronidase Type I from bovine testes (Sigma H3506 orequivalent). In an embodiment, the hyaluronidase is hyaluronidase TypeII from sheep testes (Sigma H2126 or equivalent). In an embodiment, thehyaluronidase is hyaluronidase Type III. In an embodiment, thehyaluronidase is hyaluronidase Type IV (Type IV-S) from bovine testes(Sigma H3884 or equivalent). In an embodiment, the hyaluronidase ishyaluronidase Type V from sheep testes (Sigma H6254 or equivalent). Inan embodiment, the hyaluronidase is hyaluronidase Type VIII from bovinetestes (Sigma H3757 or equivalent). In an embodiment, the hyaluronidaseis recombinant human hyaluronidase (commercially available as HYLENEXfrom Halozyme, Inc.). The preparation and properties of hyaluronidasessuitable for use in the invention are described in U.S. Pat. Nos.4,820,516; 5,593,877; 6,057,110; 6,123,938; 7,767,429; 8,202,517;8,431,124; and 8,431,380; the disclosures of each of which areincorporated by reference herein.

In an embodiment, a method of obtaining rTILs includes a step wherein atumor is digested using a deoxyribonuclease and a hyaluronidase. In anembodiment, a method of obtaining rTILs includes a step wherein a tumoris digested using a deoxyribonuclease and a collagenase. In anembodiment, a method of obtaining rTILs includes a step wherein a tumoris digested using a hyaluronidase and a collagenase. In an embodiment, amethod of obtaining rTILs includes a step wherein a tumor is digestedusing a deoxyribonuclease, a hyaluronidase, and a collagenase.

In an embodiment, a method of obtaining rTILs includes a step wherein atumor is digested using a deoxyribonuclease and a hyaluronidase and atleast one additional enzyme. In an embodiment, a method of obtainingrTILs includes a step wherein a tumor is digested using adeoxyribonuclease and a collagenase and at least one additional enzyme.In an embodiment, a method of obtaining rTILs includes a step wherein atumor is digested using a hyaluronidase and a collagenase and at leastone additional enzyme. In an embodiment, a method of obtaining rTILsincludes a step wherein a tumor is digested using a deoxyribonuclease, ahyaluronidase, and a collagenase and at least one additional enzyme. Inany of the foregoing embodiments, the additional enzyme is selected fromthe group consisting of caseinase, clostripain, trypsin, andcombinations thereof.

In an embodiment, a method of obtaining rTILs includes a step wherein atumor is digested using any enzyme described above, and furthercomprises the step of mechanically disrupting or fragmenting the tumorbefore, during, or after digestion.

In an embodiment, a method of obtaining rTILs includes a step wherein atumor is digested using any enzyme described above, wherein thedigestion is performed over a period selected from the group consistingof 15 minutes, 30 minutes, 45 minutes, 1 hour, 90 minutes, 2 hours, 3hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours,11 hours, 12 hours, 18 hours, 24 hours, 36 hours, and 48 hours.

In an embodiment, a method of obtaining rTILs includes a step wherein atumor is digested using any enzyme described above, wherein thedigestion is performed over a period selected from the group consistingof about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour,about 90 minutes, about 2 hours, about 3 hours, about 4 hours, about 5hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about10 hours, about 11 hours, about 12 hours, about 18 hours, about 24hours, about 36 hours, and about 48 hours.

In an embodiment, a method of obtaining rTILs includes a step wherein atumor is digested using any enzyme described above, wherein thedigestion is performed over a period selected from the group consistingof less than 15 minutes, less than 30 minutes, less than 45 minutes,less than 1 hour, less than 90 minutes, less than 2 hours, less than 3hours, less than 4 hours, less than 5 hours, less than 6 hours, lessthan 7 hours, less than 8 hours, less than 9 hours, less than 10 hours,less than 11 hours, less than 12 hours, less than 18 hours, less than 24hours, less than 36 hours, and less than 48 hours.

In an embodiment, a method of obtaining rTILs includes a step wherein atumor is digested using any enzyme described above, wherein thedigestion is performed over a period selected from the group consistingof greater than 15 minutes, greater than 30 minutes, greater than 45minutes, greater than 1 hour, greater than 90 minutes, greater than 2hours, greater than 3 hours, greater than 4 hours, greater than 5 hours,greater than 6 hours, greater than 7 hours, greater than 8 hours,greater than 9 hours, greater than 10 hours, greater than 11 hours,greater than 12 hours, greater than 18 hours, greater than 24 hours,greater than 36 hours, and greater than 48 hours.

In an embodiment, a method of obtaining rTILs includes a step wherein atumor is digested using any enzyme described above, wherein thedigestion is performed over a period selected from the group consistingof between 30 minutes and 1 hour, between 1 hours and 2 hours, between 2hours and 3 hours, between 3 hours and 4 hours, between 4 hours and 5hours, between 5 hours and 6 hours, between 6 hours and 12 hours,between 12 hours and 18 hours, between 18 hours and 24 hours, andbetween 24 hours and 48 hours.

In an embodiment, a method of obtaining rTILs includes a step wherein atumor is digested using any enzyme described above, wherein thedigestion is performed at a temperature selected from the groupconsisting of about 20° C., about 25° C., about 30° C., about 35° C.,about 40° C., about 45° C., about 50° C., about 55° C., about 60° C.,about 65° C., about 70° C., about 75° C., and about 80° C.

In an embodiment, a method of obtaining rTILs includes a step wherein atumor is digested using any enzyme described above, wherein thedigestion is performed at a temperature selected from the groupconsisting of between 20° C. and 25° C., between 25° C. and 30° C.,between 30° C. and 35° C., between 35° C. and 40° C., between 40° C. and45° C., between 45° C. and 50° C., between 50° C. and about 55° C.,between 55° C. and 60° C., between 60° C. and 65° C., between 65° C. and70° C., between 70° C. and 75° C., and between 75° C. and 80° C.

In an embodiment, a method of obtaining rTILs includes a step wherein atumor is digested using any enzyme described above, wherein the time andtemperature of the digestion are each decreased if tumor remnants (afterpre-REP) are digested. In an embodiment, a method of obtaining rTILsincludes a step wherein a tumor is digested using any enzyme describedabove, wherein the time and temperature of the digestion are eachincrease if whole tumor fragments (without pre-REP) are digested.

Methods of Modulating rTIL to eTIL Ratio

In an embodiment, the concentration of rTILs relative to eTILs may bemodulated or controlled by use of any expansion and digestion steps asdescribed herein (including pre-REP), such that a therapeutic TILproduct for use in the treatment of cancers described herein may containa desirable rTIL to eTIL ratio. In an embodiment, the invention providesa method of removing eTILs from a mixture of eTILs and rTILs. In anembodiment, the invention provides a method of removing rTILs from amixture of eTILs and rTILs.

In some embodiments of the methods of the invention, eTILs and/or rTILsmay be added to a culture before an initial expansion step, at a firstexpansion step (e.g., pre-REP), and/or at a second expansion step (e.g.,REP). In some embodiments of the methods of the invention, eTILS may beseparately cultured according to the culture or expansion stepsdescribed herein through one, two, three, or more expansions, and addedto a population of rTILS and eTILS at a selected rTIL to eTIL ratio. Insome embodiments of the methods of the invention, rTILS may beseparately cultured according to the culture or expansion stepsdescribed herein through one, two, three, or more expansions, and addedto a population of eTILS to provide a mixture of rTILS and eTILS at aselected rTIL to eTIL ratio.

In an embodiment, eTILs prepared according to the methods describedherein may be added to a population of rTILs to provide a selected rTILto eTIL ratio in the resulting rTIL/eTIL mixture. In an embodiment,rTILs prepared according to the methods described herein may be added toa population of eTILs to provide a selected rTIL to eTIL ratio in theresulting rTIL/eTIL mixture.

In an embodiment, the invention provides a method of treating a cancerwherein the treatment comprises delivering a therapeutically effectiveamount of TILs to a patient, wherein the ratio of rTILs to eTILs in theTILs (e.g., a selected rTIL to eTIL ratio) is selected from the groupconsisting of about 0:100, about 1:99, about 5:95, about 10:90, about15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60,about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about70:30, about 75:25, about 80:20, about 85:15, about 90:10, about 95:5,about 99:1, and about 100:0 rTIL to eTIL.

In an embodiment, the rTIL to eTIL ratio is adjusted using a selectionmethod, which may be used to enrich or reduce rTILs relative to eTILs asrequired by the skilled artisan. In an embodiment, the selection methodis based on the lack of exhaustion markers, including TIM3, LAG3, TIGIT,PD-1, and CTLA-4. In an embodiment, the selection method is based onenhanced CD69 expression. In an embodiment, the selection method isbased on superior mitochondrial mass. In an embodiment, the selectionmethod is based on a subset of cell surface proteins. In an embodiment,the selection method is based on phenotype. In an embodiment, theselection method is based on function.

In an embodiment, the rTIL to eTIL ratio is adjusted by co-culturingrTILs and eTILs in the same cell culture medium until a desirable ratiois obtained. In an embodiment, the rTIL to eTIL ratio is adjusted byco-culturing rTILs and eTILs in the same cell culture medium, includingthe addition of rTILs or eTILs to the cell culture medium at differenttimepoints during expansion, until a desirable ratio is obtained. In anembodiment, rTIL growth is preferentially expanded in the cell culturemedium by addition of cytokines other than IL-2, including IL-4, IL-7,IL-15, and/or IL-21.

In an embodiment, the ratio of rTILs to eTILs (e.g., a selected rTIL toeTIL ratio) provided by the methods described herein may be at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or99.9% rTIL to eTIL.

In an embodiment, the ratio of rTILs to eTILs (e.g., a selected rTIL toeTIL ratio) provided by the methods described herein may be at most 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or99.9% rTIL to eTIL.

In an embodiment, the ratio of rTILs to eTILs (e.g., a selected rTIL toeTIL ratio) provided by the methods described herein may be about 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or99.9% rTIL to eTIL.

Methods of Treating Cancers and Other Diseases

The rTILs and combinations of rTILs and eTILs described herein may beused in a method for treating diseases in a human. In an embodiment,they are for use in treating a hyperproliferative disorder. In someembodiments, the hyperproliferative disorder is cancer. In someembodiments, the hyperproliferative disorder is a solid tumor cancer. Insome embodiments, the solid tumor cancer is selected from the groupconsisting of melanoma, double-refractory melanoma (i.e., melanomarefractory to at least two prior treatments including chemotherapy andcheckpoint blockade), ovarian cancer, cervical cancer, non-small-celllung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancercaused by human papilloma virus, head and neck cancer, renal cancer,renal cell carcinoma, and sarcoma. In some embodiments, thehyperproliferative disorder is a hematological malignancy (or liquidtumor cancer). In some embodiments, the hematological malignancy isselected from the group consisting of acute myeloid leukemia, chroniclymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B celllymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, follicularlymphoma, and mantle cell lymphoma. The rTILs and combinations of rTILsand eTILs described herein may also be used in treating other disordersas described herein and in the following paragraphs.

In an embodiment, the invention includes a method of treating a cancerin a patient in need of such treatment, wherein the treatment comprisesdelivering a therapeutically effective amount of rTILs to a patient,wherein the rTILs are prepared according a method comprising the stepsof:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture; and    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the second cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof.

In an embodiment, the invention includes a method treating a cancer in apatient in need of such treatment, the method comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture;    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the second cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof,    -   (g) treating the patient with a non-myeloablative        lymphodepletion regimen prior to administering the rTILs to the        patient;    -   (h) administering a therapeutically effective amount of rTILs to        the patient; and    -   (i) treating the patient with a high-dose IL-2 regimen starting        on the day after administration of the rTILs to the patient.

In an embodiment, the invention includes a method treating a cancer in apatient in need of such treatment, the method comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture;    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the second cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof,    -   (g) treating the patient with a non-myeloablative        lymphodepletion regimen prior to administering the rTILs to the        patient;    -   (h) administering a therapeutically effective amount of rTILs to        the patient, wherein a therapeutically effective amount of eTILs        are simultaneously administered to the patient in a mixture with        the rTILs; and    -   (i) treating the patient with a high-dose IL-2 regimen starting        on the day after administration of the rTILs to the patient.

In an embodiment, the invention includes a method treating a cancer in apatient in need of such treatment, the method comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture;    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the second cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof,    -   (g) treating the patient with a non-myeloablative        lymphodepletion regimen prior to administering the rTILs to the        patient;    -   (h) administering a therapeutically effective amount of rTILs to        the patient, wherein a therapeutically effective amount of eTILs        are simultaneously administered to the patient in a mixture with        the rTILs; and    -   (i) treating the patient with a high-dose IL-2 regimen starting        on the day after administration of the rTILs to the patient,    -   wherein the cancer is selected from the group consisting of        melanoma, double-refractory melanoma, ovarian cancer, cervical        cancer, lung cancer, bladder cancer, breast cancer, head and        neck cancer, renal cell carcinoma, acute myeloid leukemia,        colorectal cancer, sarcoma, non-small cell lung cancer (NSCLC),        and triple negative breast cancer.

In an embodiment, the invention includes a method treating a cancer in apatient in need of such treatment, the method comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture;    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the second cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof,    -   (g) treating the patient with a non-myeloablative        lymphodepletion regimen prior to administering the rTILs to the        patient, wherein the non-myeloablative lymphodepletion regimen        comprises the steps of administration of cyclophosphamide at a        dose of 60 mg/m²/day for two days followed by administration of        fludarabine at a dose of 25 mg/m²/day for five days;    -   (h) administering a therapeutically effective amount of rTILs to        the patient, wherein a therapeutically effective amount of eTILs        are simultaneously administered to the patient in a mixture with        the rTILs; and    -   (i) treating the patient with a high-dose IL-2 regimen starting        on the day after administration of the rTILs to the patient.

In some embodiments of the methods described herein, the step ofremoving at least a plurality of the eTILs includes removing at least1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.9%, or 100% of the eTILs.

Efficacy of the compounds and combinations of compounds described hereinin treating, preventing and/or managing the indicated diseases ordisorders can be tested using various models known in the art, whichprovide guidance for treatment of human disease. For example, models fordetermining efficacy of treatments for ovarian cancer are described,e.g., in Mullany, et al., Endocrinology 2012, 153, 1585-92; and Fong, etal., J. Ovarian Res. 2009, 2, 12. Models for determining efficacy oftreatments for pancreatic cancer are described in Herreros-Villanueva,et al., World J. Gastroenterol. 2012, 18, 1286-1294. Models fordetermining efficacy of treatments for breast cancer are described,e.g., in Fantozzi, Breast Cancer Res. 2006, 8, 212. Models fordetermining efficacy of treatments for melanoma are described, e.g., inDamsky, et al., Pigment Cell & Melanoma Res. 2010, 23, 853-859. Modelsfor determining efficacy of treatments for lung cancer are described,e.g., in Meuwissen, et al., Genes & Development, 2005, 19, 643-664.Models for determining efficacy of treatments for lung cancer aredescribed, e.g., in Kim, Clin. Exp. Otorhinolaryngol. 2009, 2, 55-60;and Sano, Head Neck Oncol. 2009, 1, 32.

Co-Administration of IL-2

In an embodiment, the invention provides a method of treating a cancerin a patient in need of such treatment, comprising the steps of:

-   -   (a) obtaining rTILs from a tumor resected from a patient        according to a method described herein;    -   (b) treating the patient with a non-myeloablative        lymphodepletion regimen prior to administering the rTILs to the        patient;    -   (c) administering a therapeutically effective amount of rTILs to        the patient; and    -   (d) treating the patient with an IL-2 regimen starting on the        day after administration of the rTILs to the patient.

In an embodiment, the IL-2 regimen comprises a high-dose IL-2 regimen,wherein the high-dose IL-2 regimen comprises aldesleukin, or abiosimilar or variant thereof, administered intravenously starting onthe day after administering a therapeutically effective portion of thethird population of TILs, wherein the aldesleukin or a biosimilar orvariant thereof is administered at a dose of 600,000 or 720,000 IU/kg(patient body mass) using 15-minute bolus intravenous infusions everyeight hours until tolerance, for a maximum of 14 doses. Following 9 daysof rest, this schedule may be repeated for another 14 doses, for amaximum of 28 doses in total.

In an embodiment, the IL-2 regimen comprises a high-dose IL-2 regimen,wherein the high-dose IL-2 regimen comprises aldesleukin, or abiosimilar or variant thereof, administered intravenously starting onthe day after administering a therapeutically effective portion of thethird population of TILs, wherein the aldesleukin or a biosimilar orvariant thereof is administered at a dose of 0.037 mg/kg or 0.044 mg/kgIU/kg (patient body mass) using 15-minute bolus intravenous infusionsevery eight hours until tolerance, for a maximum of 14 doses. Following9 days of rest, this schedule may be repeated for another 14 doses, fora maximum of 28 doses in total.

In an embodiment, the invention includes a method treating a cancer in apatient in need of such treatment, the method comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture;    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the second cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof,    -   (g) treating the patient with a non-myeloablative        lymphodepletion regimen prior to administering the rTILs to the        patient;    -   (h) administering a therapeutically effective amount of rTILs to        the patient, wherein a therapeutically effective amount of eTILs        are simultaneously administered to the patient in a mixture with        the rTILs; and    -   (i) treating the patient with a high-dose IL-2 regimen starting        on the day after administration of the rTILs to the patient,    -   wherein the high-dose IL-2 regimen comprises 600,000 or 720,000        IU/kg of aldesleukin, or a biosimilar or variant thereof,        administered as a 15-minute bolus intravenous infusion every        eight hours until tolerance.

In an embodiment, the IL-2 regimen comprises a decrescendo IL-2 regimen.Decrescendo IL-2 regimens have been described in O'Day, et al., J. Clin.Oncol. 1999, 17, 2752-61 and Eton, et al., Cancer 2000, 88, 1703-9, thedisclosures of which are incorporated herein by reference. In anembodiment, a decrescendo IL-2 regimen comprises 18×10⁶ IU/m²administered intravenously over 6 hours, followed by 18×10⁶ IU/m²administered intravenously over 12 hours, followed by 18×10⁶ IU/m²administered intravenously over 24 hours, followed by 4.5×10⁶ IU/m²administered intravenously over 72 hours. This treatment cycle may berepeated every 28 days for a maximum of four cycles. In an embodiment, adecrescendo IL-2 regimen comprises 18,000,000 IU/m² on day 1, 9,000,000IU/m² on day 2, and 4,500,000 IU/m² on days 3 and 4.

In an embodiment, the invention includes a method treating a cancer in apatient in need of such treatment, the method comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture;    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the second cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof,    -   (g) treating the patient with a non-myeloablative        lymphodepletion regimen prior to administering the rTILs to the        patient;    -   (h) administering a therapeutically effective amount of rTILs to        the patient, wherein a therapeutically effective amount of eTILs        are simultaneously administered to the patient in a mixture with        the rTILs; and    -   (i) treating the patient with a decrescendo IL-2 regimen        starting on the day after administration of the rTILs to the        patient,    -   wherein the decrescendo IL-2 regimen comprises 18×10⁶ IU/m²        administered intravenously over 6 hours, followed by 18×10⁶        IU/m² administered intravenously over 12 hours, followed by        18×10⁶ IU/m² administered intravenously over 24 hours, followed        by 4.5×10⁶ IU/m² administered intravenously over 72 hours,        repeated every 28 days for a maximum of four cycles.

In an embodiment, the IL-2 regimen comprises administration of pegylatedIL-2, including pegylated aldesleukin. In an embodiment, the IL-2regimen comprises administration of pegylated IL-2 every 1, 2, 4, 6, 7,14 or 21 days at a dose of 0.10 mg/day to 50 mg/day.

In an embodiment, the invention includes a method treating a cancer in apatient in need of such treatment, the method comprising:

-   -   (a) obtaining tumor tissue from the patient, wherein the tumor        tissue comprises tumor infiltrating lymphocytes (TILs);    -   (b) fragmenting the tumor tissue;    -   (c) treating the tumor tissue in a gas permeable container with        a first cell culture medium and interleukin 2 (IL-2) to provide        tumor remnants and emergent TILs (eTILs);    -   (d) removing at least a plurality of the eTILs;    -   (e) enzymatically digesting the tumor remnants into tumor        remnant cells using a digest mixture;    -   (f) expanding the tumor remnant cells with a second cell culture        medium comprising cell culture media, irradiated feeder cells,        OKT-3 antibody, and IL-2 in a gas permeable container to provide        an expanded number of remnant tumor infiltrating lymphocytes        (rTILs);    -   wherein the rTILs express reduced levels of a T cell exhaustion        marker relative to the eTILs,    -   wherein the T cell exhaustion marker is selected from the group        consisting of TIM3, LAG3, TIGIT, PD-1, and combinations thereof,        and    -   wherein the second cell culture medium further comprises a        cytokine selected from the group consisting of IL-4, IL-7,        IL-15, IL-21, and combinations thereof,    -   (g) treating the patient with a non-myeloablative        lymphodepletion regimen prior to administering the rTILs to the        patient;    -   (h) administering a therapeutically effective amount of rTILs to        the patient, wherein a therapeutically effective amount of eTILs        are simultaneously administered to the patient in a mixture with        the rTILs; and    -   (i) treating the patient with a pegylated IL-2 regimen starting        on the day after administration of the rTILs to the patient,    -   wherein the pegylated IL-2 regimen comprises administration of        pegylated IL-2 every 1, 2, 4, 6, 7, 14 or 21 days at a dose of        0.10 mg/day to 50 mg/day.        Non-Myeloablative Lymphodepletion with Chemotherapy

In an embodiment, the invention includes a method of treating a cancerwith a population of rTILs, wherein a patient is pre-treated withnon-myeloablative chemotherapy prior to an infusion of rTILs accordingto the invention. In some embodiments, the population of rTILs may beprovided with a population of eTils, wherein a patient is pre-treatedwith non-myeloablative chemotherapy prior to an infusion of rTILs andeTils according to the invention. In an embodiment, thenon-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/d for 2 days(days 27 and 26 prior to rTIL infusion) and fludarabine 25 mg/m²/d for 5days (days 27 to 23 prior to rTIL infusion). In an embodiment, afternon-myeloablative chemotherapy and rTIL infusion (at day 0) according tothe invention, the patient receives an intravenous infusion of IL-2intravenously at 720,000 IU/kg every 8 hours to physiologic tolerance.

Experimental findings indicate that lymphodepletion prior to adoptivetransfer of tumor-specific T lymphocytes plays a key role in enhancingtreatment efficacy by eliminating regulatory T cells and competingelements of the immune system (“cytokine sinks”). Accordingly, someembodiments of the invention utilize a lymphodepletion step (sometimesalso referred to as “immunosuppressive conditioning”) on the patientprior to the introduction of the rTILs of the invention.

In general, lymphodepletion is achieved using administration offludarabine or cyclophosphamide (the active form being referred to asmafosfamide) and combinations thereof. Such methods are described inGassner, et al., Cancer Immunol. Immunother. 2011, 60, 75-85, Muranski,et al., Nat. Clin. Pract. Oncol., 2006, 3, 668-681, Dudley, et al., J.Clin. Oncol. 2008, 26, 5233-5239, and Dudley, et al., J. Clin. Oncol.2005, 23, 2346-2357, all of which are incorporated by reference hereinin their entireties.

In some embodiments, the fludarabine is administered at a concentrationof 0.5 μg/mL-10 μg/mL fludarabine. In some embodiments, the fludarabineis administered at a concentration of 1 μg/mL fludarabine. In someembodiments, the fludarabine treatment is administered for 1 day, 2days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In someembodiments, the fludarabine is administered at a dosage of 10mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 25 mg/kg/day, 30 mg/kg/day, 35mg/kg/day, 40 mg/kg/day, or 45 mg/kg/day. In some embodiments, thefludarabine treatment is administered for 2-7 days at 35 mg/kg/day. Insome embodiments, the fludarabine treatment is administered for 4-5 daysat 35 mg/kg/day. In some embodiments, the fludarabine treatment isadministered for 4-5 days at 25 mg/kg/day.

In some embodiments, the mafosfamide, the active form ofcyclophosphamide, is obtained at a concentration of 0.5 μg/mL-10 μg/mLby administration of cyclophosphamide. In some embodiments, mafosfamide,the active form of cyclophosphamide, is obtained at a concentration of 1μg/mL by administration of cyclophosphamide. In some embodiments, thecyclophosphamide treatment is administered for 1 day, 2 days, 3 days, 4days, 5 days, 6 days, or 7 days or more. In some embodiments, thecyclophosphamide is administered at a dosage of 100 mg/m²/day, 150mg/m²/day, 175 mg/m²/day, 200 mg/m²/day, 225 mg/m²/day, 250 mg/m²/day,275 mg/m²/day, or 300 mg/m²/day. In some embodiments, thecyclophosphamide is administered intravenously (i.v.) In someembodiments, the cyclophosphamide treatment is administered for 2-7 daysat 35 mg/kg/day. In some embodiments, the cyclophosphamide treatment isadministered for 4-5 days at 250 mg/m²/day i.v. In some embodiments, thecyclophosphamide treatment is administered for 4 days at 250 mg/m²/dayi.v.

In some embodiments, lymphodepletion is performed by administering thefludarabine and the cyclophosphamide are together to a patient. In someembodiments, fludarabine is administered at 25 mg/m²/day i.v. andcyclophosphamide is administered at 250 mg/m²/day i.v. over 4 days.

In an embodiment, the lymphodepletion is performed by administration ofcyclophosphamide at a dose of 60 mg/m²/day for two days followed byadministration of fludarabine at a dose of 25 mg/m²/day for five days.

Pharmaceutical Compositions, Dosages, and Dosing Regimens

In an embodiment, rTILs expanded using methods of the invention areadministered to a patient as a pharmaceutical composition. In anembodiment, the pharmaceutical composition is a suspension of rTILs in asterile buffer. rTILs expanded using methods of the invention may beadministered by any suitable route as known in the art. Preferably, therTILs are administered as a single intra-arterial or intravenousinfusion, which preferably lasts approximately 30 to 60 minutes. Othersuitable routes of administration include intraperitoneal, intrathecal,and intralymphatic administration.

In an embodiment, rTILs and eTILs expanded using methods of theinvention are administered to a patient as a pharmaceutical composition.In an embodiment, the pharmaceutical composition is a suspension ofrTILs and eTILs in a sterile buffer. rTILs and eTILs expanded usingmethods of the invention may be administered by any suitable route asknown in the art. Preferably, the rTILs and eTILs are administered as asingle intra-arterial or intravenous infusion, which preferably lastsapproximately 30 to 60 minutes. Other suitable routes of administrationinclude intraperitoneal, intrathecal, and intralymphatic administration.

Any suitable dose of rTILs can be administered. Preferably, from about2.3×10¹⁰ to about 13.7×10¹⁰ rTILs are administered, with an average ofaround 7.8×10¹⁰ rTILs, particularly if the cancer is melanoma. In anembodiment, about 1.2×10¹⁰ to about 4.3×10¹⁰ of rTILs are administered.

Any suitable dose of rTILs and eTILs can be administered. Preferably,from about 2.3×10¹⁰ to about 13.7×10¹⁰ rTILs and eTILs are administered,with an average of around 7.8×10¹⁰ rTILs and eTILs, particularly if thecancer is melanoma. In an embodiment, about 1.2×10¹⁰ to about 4.3×10¹⁰of rTILs and eTILs are administered.

In some embodiments, the number of the rTILs provided in thepharmaceutical compositions of the invention is about 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰,6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹,6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 2×10¹², 3×10¹², 4×10¹², 5×10¹²,6×10¹², 7×10¹², 8×10¹², 9×10¹², 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³, 5×10¹³,6×10¹³, 7×10¹³, 8×10¹³, and 9×10¹³. In an embodiment, the number of therTILs provided in the pharmaceutical compositions of the invention is inthe range of 1×10⁶ to 5×10⁶, 5×10⁶ to 1×10⁷, 1×10⁷ to 5×10⁷, 5×10⁷ to1×10⁸, 1×10⁸ to 5×10⁸, 5×10⁸ to 1×10⁹, 1×10⁹ to 5×10⁹, 5×10⁹ to 1×10¹⁰,1×10¹⁰ to 5×10¹⁰, 5×10¹⁰ to 1×10¹¹, 5×10¹¹ to 1×10¹², 1×10¹² to 5×10¹²,and 5×10¹² to 1×10¹³.

In some embodiments, the number of the rTILs and eTILs provided in thepharmaceutical compositions of the invention is about 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰,6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹,6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 2×10¹², 3×10¹², 4×10¹², 5×10¹²,6×10¹², 7×10¹², 8×10¹², 9×10¹², 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³, 5×10¹³,6×10¹³, 7×10¹³, 8×10¹³, and 9×10¹³. In an embodiment, the number of therTILs and eTILs provided in the pharmaceutical compositions of theinvention is in the range of 1×10⁶ to 5×10⁶, 5×10⁶ to 1×10⁷, 1×10⁷ to5×10⁷, 5×10⁷ to 1×10⁸, 1×10⁸ to 5×10⁸, 5×10⁸ to 1×10⁹, 1×10⁹ to 5×10⁹,5×10⁹ to 1×10¹⁰, 1×10¹⁰ to 5×10¹⁰, 5×10¹⁰ to 1×10¹¹, 5×10¹¹ to 1×10¹²,1×10¹² to 5×10¹², and 5×10¹² to 1×10¹³.

In some embodiments, the concentration of the rTILs provided in thepharmaceutical compositions of the invention is less than, for example,100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%,14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%,0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%,0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%,0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%,0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v of the pharmaceuticalcomposition.

In some embodiments, the concentration of the rTILs and eTILs providedin the pharmaceutical compositions of the invention is less than, forexample, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%,16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%,0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%,0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%,0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v of thepharmaceutical composition.

In some embodiments, the concentration of the rTILs provided in thepharmaceutical compositions of the invention is greater than 90%, 80%,70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%,18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25%16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%,13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25%11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%,8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%,5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%,2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%,0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%,0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%,0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001%w/w, w/v, or v/v of the pharmaceutical composition.

In some embodiments, the concentration of the rTILs and eTILs providedin the pharmaceutical compositions of the invention is greater than 90%,80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%,18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25%16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25%, 14%, 13.75%,13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25%, 12%, 11.75%, 11.50%,11.25%, 11%, 10.75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%, 9.25%, 9%,8.75%, 8.50%, 8.25%, 8%, 7.75%, 7.50%, 7.25%, 7%, 6.75%, 6.50%, 6.25%,6%, 5.75%, 5.50%, 5.25%, 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%,3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%,0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%,0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%,0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%,0.0003%, 0.0002% or 0.0001%, w/w, w/v, or v/v of the pharmaceuticalcomposition.

In some embodiments, the concentration of the rTILs provided in thepharmaceutical compositions of the invention is in the range from about0.0001%, to about 50%, about 0.001%, to about 40%, about 0.01%, to about30%, about 0.02%, to about 29%, about 0.03%, to about 28%, about 0.04%,to about 27%, about 0.05%, to about 26%, about 0.06%, to about 25%,about 0.07%, to about 24%, about 0.08%, to about 23%, about 0.09%, toabout 22%, about 0.1%, to about 21%, about 0.2% to about 20%, about 0.3%to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%,about 0.9%, to about 12% or about 1%, to about 10% w/w, w/v or v/v ofthe pharmaceutical composition.

In some embodiments, the concentration of the rTILs and eTILs providedin the pharmaceutical compositions of the invention is in the range fromabout 0.0001% to about 50%, about 0.001%, to about 40%, about 0.01%, toabout 30%, about 0.02%, to about 29%, about 0.03%, to about 28%, about0.04%, to about 27%, about 0.05% to about 26%, about 0.06%, to about25%, about 0.07%, to about 24%, about 0.08%, to about 23%, about 0.09%,to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%,about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about14%, about 0.9%, to about 12% or about 1% to about 10% w/w, w/v or v/vof the pharmaceutical composition.

In some embodiments, the concentration of the rTILs provided in thepharmaceutical compositions of the invention is in the range from about0.001% to about 10%, about 0.01% to about 5%, about 0.02%, to about4.5%, about 0.03%, to about 4%, about 0.04%, to about 3.5%, about 0.05%to about 3%, about 0.06%, to about 2.5%, about 0.07%, to about 2%, about0.08%, to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9%w/w, w/v or v/v of the pharmaceutical composition.

In some embodiments, the concentration of the rTILs and eTILs providedin the pharmaceutical compositions of the invention is in the range fromabout 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% toabout 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9%w/w, w/v or v/v of the pharmaceutical composition.

In some embodiments, the amount of the rTILs provided in thepharmaceutical compositions of the invention is equal to or less than 10g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g,4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g,0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g,0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g,0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g, or0.0001 g.

In some embodiments, the amount of the rTILs and eTILs provided in thepharmaceutical compositions of the invention is equal to or less than 10g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g,4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g,0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g,0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g,0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g, or0.0001 g.

In some embodiments, the amount of the rTILs provided in thepharmaceutical compositions of the invention is more than 0.0001 g,0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g,0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g,0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g,0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g,0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or10 g.

In some embodiments, the amount of the rTILs and eTILs provided in thepharmaceutical compositions of the invention is more than 0.0001 g,0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g,0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g,0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g,0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g,0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or10 g.

The rTILs provided in the pharmaceutical compositions of the inventionare effective over a wide dosage range. The exact dosage will dependupon the route of administration, the form in which the compound isadministered, the gender and age of the subject to be treated, the bodyweight of the subject to be treated, and the preference and experienceof the attending physician. The clinically-established dosages of therTILs may also be used if appropriate. The amounts of the pharmaceuticalcompositions administered using the methods herein, such as the dosagesof rTILs, will be dependent on the human or mammal being treated, theseverity of the disorder or condition, the rate of administration, thedisposition of the active pharmaceutical ingredients and the discretionof the treating physician.

The rTILs and eTILs provided in the pharmaceutical compositions of theinvention are effective over a wide dosage range. The exact dosage willdepend upon the route of administration, the form in which the compoundis administered, the gender and age of the subject to be treated, thebody weight of the subject to be treated, and the preference andexperience of the attending physician. The clinically-establisheddosages of the rTILs and eTILs may also be used if appropriate. Theamounts of the pharmaceutical compositions administered using themethods herein, such as the dosages of rTILs and eTILs, will bedependent on the human or mammal being treated, the severity of thedisorder or condition, the rate of administration, the disposition ofthe active pharmaceutical ingredients and the discretion of the treatingphysician.

In some embodiments, rTILs may be administered in a single dose. Suchadministration may be by injection, e.g., intravenous injection. In someembodiments, rTILs may be administered in multiple doses. Dosing may beonce, twice, three times, four times, five times, six times, or morethan six times per year. Dosing may be once a month, once every twoweeks, once a week, or once every other day. Administration of rTILs maycontinue as long as necessary.

In some embodiments, rTILs and eTILs may be administered in a singledose. Such administration may be by injection, e.g., intravenousinjection. In some embodiments, rTILs may be administered in multipledoses. Dosing may be once, twice, three times, four times, five times,six times, or more than six times per year. Dosing may be once a month,once every two weeks, once a week, or once every other day.Administration of rTILs and eTILs may continue as long as necessary.

In some embodiments, an effective dosage of rTILs is about 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰,6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹,6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 2×10¹², 3×10¹², 4×10¹², 5×10¹²,6×10¹², 7×10¹², 8×10¹², 9×10¹², 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³, 5×10¹³,6×10¹³, 7×10¹³, 8×10¹³, and 9×10¹³. In some embodiments, an effectivedosage of rTILs is in the range of 1×10⁶ to 5×10⁶, 5×10⁶ to 1×10⁷, 1×10⁷to 5×10⁷, 5×10⁷ to 1×10⁸, 1×10⁸ to 5×10⁸, 5×10⁸ to 1×10⁹, 1×10⁹ to5×10⁹, 5×10⁹ to 1×10¹⁰, 1×10¹⁰ to 5×10¹⁰, 5×10¹⁰ to 1×10¹¹, 5×10¹¹ to1×10¹², 1×10¹² to 5×10¹², and 5×10¹² to 1×10¹³.

In some embodiments, an effective dosage of rTILs and eTILs is about1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷,2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸,3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹,4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰,4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹,4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 2×10¹², 3×10¹²,4×10¹², 5×10¹², 6×10¹², 7×10¹², 8×10¹², 9×10¹², 1×10¹³, 2×10¹³, 3×10¹³,4×10¹³, 5×10¹³, 6×10¹³, 7×10¹³, 8×10¹³, and 9×10¹³. In some embodiments,an effective dosage of rTILs and eTILs is in the range of 1×10⁶ to5×10⁶, 5×10⁶ to 1×10⁷, 1×10⁷ to 5×10⁷, 5×10⁷ to 1×10⁸, 1×10⁸ to 5×10⁸,5×10⁸ to 1×10⁹, 1×10⁹ to 5×10⁹, 5×10⁹ to 1×10¹⁰, 1×10¹⁰ to 5×10¹⁰,5×10¹⁰ to 1×10¹¹, 5×10¹¹ to 1×10¹², 1×10¹² to 5×10¹², and 5×10¹² to1×10¹³.

In some embodiments, an effective dosage of rTILs is in the range ofabout 0.01 mg/kg to about 4.3 mg/kg, about 0.15 mg/kg to about 3.6mg/kg, about 0.3 mg/kg to about 3.2 mg/kg, about 0.35 mg/kg to about2.85 mg/kg, about 0.15 mg/kg to about 2.85 mg/kg, about 0.3 mg to about2.15 mg/kg, about 0.45 mg/kg to about 1.7 mg/kg, about 0.15 mg/kg toabout 1.3 mg/kg, about 0.3 mg/kg to about 1.15 mg/kg, about 0.45 mg/kgto about 1 mg/kg, about 0.55 mg/kg to about 0.85 mg/kg, about 0.65 mg/kgto about 0.8 mg/kg, about 0.7 mg/kg to about 0.75 mg/kg, about 0.7 mg/kgto about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg toabout 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kgmg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg, or about 2.85mg/kg to about 2.95 mg/kg.

In some embodiments, an effective dosage of rTILs and eTILs is in therange of about 0.01 mg/kg to about 4.3 mg/kg, about 0.15 mg/kg to about3.6 mg/kg, about 0.3 mg/kg to about 3.2 mg/kg, about 0.35 mg/kg to about2.85 mg/kg, about 0.15 mg/kg to about 2.85 mg/kg, about 0.3 mg to about2.15 mg/kg, about 0.45 mg/kg to about 1.7 mg/kg, about 0.15 mg/kg toabout 1.3 mg/kg, about 0.3 mg/kg to about 1.15 mg/kg, about 0.45 mg/kgto about 1 mg/kg, about 0.55 mg/kg to about 0.85 mg/kg, about 0.65 mg/kgto about 0.8 mg/kg, about 0.7 mg/kg to about 0.75 mg/kg, about 0.7 mg/kgto about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg toabout 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kgmg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg, or about 2.85mg/kg to about 2.95 mg/kg.

In some embodiments, an effective dosage of rTILs is in the range ofabout 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg toabout 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg,about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg toabout 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg,about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg toabout 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg,or about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg,about 195 mg to about 205 mg, or about 198 to about 207 mg.

In some embodiments, an effective dosage of rTILs and eTILs is in therange of about 1 mg to about 500 mg, about 10 mg to about 300 mg, about20 mg to about 250 mg, about 25 mg to about 200 mg, about 1 mg to about50 mg, about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15mg to about 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about110 mg, or about 95 mg to about 105 mg, about 98 mg to about 102 mg,about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mgto about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210mg, about 195 mg to about 205 mg, or about 198 to about 207 mg.

An effective amount of the rTILs and/or eTILs may be administered ineither single or multiple doses by any of the accepted modes ofadministration of agents having similar utilities, including by infusioninto the bloodstream, infusion into a tumor, intra-arterial injection,intravenously, intraperitoneally, parenterally, intramuscularly,subcutaneously, topically, by intranasal administration, bytransplantation, or by inhalation.

EXAMPLES

The embodiments encompassed herein are now described with reference tothe following examples. These examples are provided for the purpose ofillustration only and the disclosure encompassed herein should in no waybe construed as being limited to these examples, but rather should beconstrued to encompass any and all variations which become evident as aresult of the teachings provided herein.

Example 1—Expansion of rTILs from Tumor Digests

Tumor remnants were digested according to the following exemplaryprocedure. This procedure describes the digestion of a fresh human tumorsample into a viable, single-cell suspension, to obtain and isolatetumor-infiltrating lymphocytes, and may useDNase-Collagenase-Hyaluronidase (DCH) methods (as described herein) orMACS human tumor dissociation kit (TDK) (Miltenyi Biotech, Inc., SanDiego, Calif., USA) digestion protocols for the dissociation of humantumors.

Preparation of the CM1+IL-2 working culture medium is as follows. Place500 mL RPMI 1640, 200 mM L-glutamine, and 100 mL human AB serum in awater bath at 37° C. to equilibrate for at least 30 minutes. Transferthe contents of this mixture from the water bath to a biosafety cabinetalong with 1000× ß-ME stock and 50 mg/mL gentamicin stock solution fromthe refrigerator. Remove 50 mL from the RPMI 1640, add: 50 mL human ABserum, 5 mL 200 mM L-glutamine, 500 μL 1000× ß-ME, and 500 μL 50 mg/mLgentamicin. To complete medium 1, add 500 μL of 6000 U/mL reconstitutedhuman rhIL-2 (CellGenix, Inc., Portsmouth, N.H., USA).

The 10×DCH stock solution is prepared using the following procedure,which is depicted in FIG. 1. First, the volume required to reconstituteeach enzyme to obtain the desired working solution concentrations iscalculated. For example, reconstitute 150,000 U (international units) ofdeoxyribonuclease in 15 mL to obtain a 10,000 U/mL working solution.Aliquot leftover working solution. Reconstitute the lyophilized enzymesin an amount of sterile Hanks' balanced salt solution (HBSS, SigmaH6648, Sigma-Aldrich Co., St. Louis, Mo., USA, or equivalent) previouslycalculated above at room temperature. Remove any residual powder fromthe sides of the bottles and from the protective foil. Pipette up anddown several times and swirl to ensure complete reconstitution. Add100,000 U of DNase (deoxyribonuclease I from bovine pancreas, SigmaD5025 or equivalent), 1 g of collagenase (Sigma C5138 from Clostridiumhistolyticum or equivalent), and 100 mg of hyaluronidase (Type V fromsheep testes, Sigma H6254 or equivalent) to a final volume of 100 mLsterile HBSS to obtain a 10× triple enzyme digestion stock solution forhuman tumors. Aliquot the remaining enzyme working solutions into 10,000U/mL DNase, 10 mg/mL collagenase and 1 mg/mL hyaluronidase. The 10×DCHstock solution at 100 mL final volume has the following concentrations:DNase I 1000 U/mL, collagenase 10 mg/mL, and hyaluronidase 1 mg/mL. The10×DCH stock solution is diluted to 1×DCH in HBSS for tumor digestion.

Concurrently, for comparison of the DCH digest with MACS TDK, preparethe reagents included in the MACS TDK to manufacturer specifications, ifdesired. Thaw aliquots that have been stored at −20° C. at roomtemperature.

The tumor may be prepared for digestion as follows. Remove the tumorfrom its primary and secondary packaging and weigh the vial, record themass, and transfer to a biosafety cabinet. Cut the tumor into fragments,or morcellate the tumor. Several fragments are selected to be used inthe digestion protocol, and additional fragments are retained forhistology and DNA extractions if desired.

An exemplary DCH-based tumor digestion procedure is depicted in FIG. 2and includes the following steps. The 10×DCH stock solution must bediluted to a 1× working concentration for digestions. Calculate thetotal volume needed for the digestion of the tumor, which is about 5 mLof solution per cm² of tumor. Dilute the DCH working solution to 1× byadding 1 part DCH to 9 parts HBSS. Transfer tumor fragments to a 50 mLFlacon conical tube in the volume of HBSS calculated above. Add theamount of 10×DCH calculated above, cap the tube, and optionally seal.Transfer to the MACS tube rotator (Miltenyi Biotech, Inc., San Diego,Calif., USA) in a 37° C., 5% CO₂ humidified incubator on constantrotation for 1 to 2 hours. Alternatively, the tumor fragments can bedigested at room temperature overnight, also with constant rotation.Attach a 0.70 μm strainer to sterile Falcon conical tube. Obtain thedigestion from the incubator and using a pipette, and add all contentsof the digestion to the strainer. Use the butt of a sterile syringeplunger to push any solid through the strainer. The tube is capped andcontains DCH-digested rTILs. The cells may be washed to remove thedigest cocktail, counted, and resuspended in media for REP expansion asdescribed elsewhere herein.

If a pre-REP step is desired to provide eTILs for comparison with rTILs(as in the following Examples), seed G-REX flasks for pre-REP using theDCH-digested rTILs. Label the necessary number of G-REX 10 flasks andadd the digest. Add CM1+IL-2 to obtain 40 mL final volume. Place theflasks in a 5% CO₂ incubator at 37° C. with humidity. Cell counting andviability may be performed using a Nexcelom Cellometer K2 using 40 μL ofsample to 40 μL of acrydine orange and propidium iodide dual stainingsolution (AOPI) solution, and count in duplicate for each digest orcondition, diluting as needed. Mix samples well to avoid clumping, andpipette AOPI promptly before running each sample to ensure viabilityisn't obfuscated by the cytotoxic effect of propidium iodide.

The DCH procedure described above was found to be surprisingly superiorto the MACS TDK enzymatic digest mixture and procedure for severalreasons. Three independent experiments using the MACS TDK mixture wereperformed. The first experiment was performed using a melanoma tumor,where a significant downregulation of the CD4⁺/CD8⁺ population in rTILswas observed by flow cytometry using the MACS system. Such an effect wasnot observed in DCH-digested rTILs, indicating that the MACS digestprocedure may adversely affect the expression of surface markers. In asecond experiment, an estrogen receptor-positive (ER+)/progesteronereceptor-positive (PR+) breast tumor was used, and the MACS digest letto the appearance of debris in the 24-well G-REX plate, whereas the DCHdigest did led to clear material, indicating poor digestion for the MACSenzyme cocktail. Finally, a second digest of a different ER+/PR+ breasttumor using the MACS TDK enzymatic digest mixture and procedure led toboth poor yield and viability of rTILs.

Example 2—Phenotypic Characterization of rTILs from Tumor Digests

During the pre-REP, tumor-resident TILs emigrate as eTILs andproliferate. The length of the pre-REP used to prepare eTILs forcomparison with rTILs may vary between 11-21 days, depending on cellgrowth. Residual tumor fragments (remnants) are normally discarded andthe expanded eTIL are subjected to a REP with irradiated PBMC feeders,anti-CD3 and IL-2. Viable TILs remaining in the tumor remnants (rTILs)following the pre-REP were investigated after digestion according toExample 1 as described above to assess their function and phenotype incomparison to eTILs.

Cell populations from the tumor remnants and pre-REP suspension (i.e.,the expanded cell population) in melanoma, head and neck, breast, renal,pancreatic, lung and colorectal tumors (n=17) were evaluated andcompared. Interestingly, rTILs are consistently phenotypically distinctfrom eTILs, as determined herein and shown by differential expression ofvarious markers including LAG3, TIM-3, PD-1, CD69, CD45RO, CD27, CD56,CD57 and HLA-DR. A REP of the tumor remnant and pre-REP populationsresulted in comparable expansion, but similar to the pre-REP results,the phenotypic signature varied between the two populations with respectto LAG3, TIM-3, HLA-DR and CD28.

The rTIL and eTIL obtained from melanoma, breast, renal, pancreatic,lung and colorectal tumors (n=9) were evaluated and compared. Tumor rTILare consistently phenotypically distinct from eTIL, as determined bydifferential expression of various markers (Table 3 and FIG. 3).

TABLE 3 Summary of phenotypic characterization results for nine tumors.LAG3 TIM3 PD-1 CD69 CD154 CD28 CD57 (CD8⁺/ (CD8⁺/ (CD8⁺/ (CD8⁺/ (CD8⁺/(CD8⁺/ (CD8⁺/ Marker CD4⁺) CD4⁺) CD4⁺) CD4⁺) CD4⁺) CD4⁺) CD4⁺) CD56Expression MFI MFI % MFI MFI MFI % % eTIL 507/144 2832/1756 36.95/47 1320/1543  1498/3751 1163/5036 18.76/19.6 5.615 rTIL 209/106 877/74242.8/48 3437/223.4 1034/1167 458.3/2795  9.16/8.5 1.027 *P-values0.05/0.21 0.05/0.01  0.38/0.89  0.11/0.001 0.55/0.01 0.05/0.11 0.05/0.06 0.05 (CD8/CD4) *P-values represent the difference betweenrTIL and eTIL using students unpaired T test.

The fundamental differences in rTILs as compared to eTILs were increasedCD69 expression (7-fold median fluorescence intensity (MFI) in CD4⁺)(≤0.0001), diminished LAG3 expression (2-fold MFI in CD8⁺ T cells)(p<0.05) and TIM3 expression (3- and 2-fold MFI in CD8⁺ and CD4⁺ Tcells, respectively) (p<0.05/0.01), diminished CD154 expression (3-foldMFI in CD4⁺ T cells) (p<0.01), and diminished CD56 expression (5%)(p<0.05). Surprisingly, a REP of rTILs and eTILs resulted in comparableexpansion, similar to the pre-REP results, as described in Example 4.The phenotypic signature of rTILs was sustained after REP with fidelitywith of expression to the individual levels of LAG3, TIM3, and CD28, asdescribed in Example 4. Furthermore, since CD57 is a receptor associatedwith terminal differentiation, the results suggest that rTILs are lessterminally differentiated than eTILs (i.e., less likely to die).

The results reported in FIG. 3 and Table 3 show that eTIL and rTILexhibit distinct yet consistent differences in phenotypic expression invarious tumor histologies. Most notably, there was a reduction in theexpression of the so called “exhaustion markers” (LAG3 and TIM3) in therTIL. Interestingly, PD-1 was similarly expressed in the eTIL and rTIL.Additionally, there was an enhancement in CD69 expression in the rTIL,compared to the eTIL, yet KLRG1 was similar between the two populations(data not shown). This provides further evidence that the rTIL do notappear to be terminally differentiated, but phenotypically resembletissue-resident effector memory T cells.

Collectively, these results have identified significant differences inthe biology of cell populations that remain in the tumor or expand andprogress out of the tumor, and the signals associated with emigrationand retention.

Example 3—Functional Characterization of rTILs from Tumor Digests

T cell dysfunction is directly associated with a loss of mitochondrialfunction. Sharping, et al., Immunity 2016, 45, 374-88. Moreover, thereprogramming of T cells to favor mitochondrial biogenesis can increaseintratumoral T cell persistence and function. Therefore, moremetabolically active T cells are pivotal in mounting an efficient immuneresponse to tumor. In an effort to assess the eTIL and rTILfunctionally, eTIL and rTIL were compared in terms of metabolic capacityvia Mitotracker and2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG).2-NBDG may be used to measure glucose uptake, but does not specify theprimary metabolic process; i.e., full oxidation in the mitochondria oronly glycolysis and generation of lactate. Mitotracker dye (ThermoFisherScientific, Inc., Waltham, Mass., USA) may be used to measuremitochondrial mass. Comparison of the eTIL and rTIL by this approachdemonstrated an enhancement in glucose update in the rTIL, as shown inFIG. 4. This result is surprising because rTILs, being directlyliberated from the tumor, are expected to be more glycolytic; however,when the mitochondrial mass the rTILs was assessed, they exhibited aslightly enhanced level of Mitotracker compared to the eTIL. Theseresults demonstrate that rTIL were more metabolically active than eTIL,when expected to be less active, and suggest that the rTIL may have agreater capacity to amount an immune response to tumor than eTIL.

To further assess their functional capabilities, rTILs were stimulatedovernight with brefeldin A and beads coated with anti-CD3, anti-CD28,and anti-CD137 antibodies (DYNABEADS, catalog no. 11162D, commerciallyavailable from ThermoFisher Scientific, Inc., Waltham, Mass., USA) andIFN-γ was measured. Cells were harvested and stained intracellularly forIFN-γ following permeabilization and assessed by flow cytometry. Theresults are shown in FIG. 5.

Cells were also stimulated with phorbol 12-myristate 13-acetate (PMA)and ionomycin to evaluate the capability of TILs to produce cytokine.The results are also shown in FIG. 5. Granzyme B, TNF-α and IL-17Alevels were also assessed. There was negligible IL-17A, and nodifferences in TNFα or granzyme B between rTILs and eTILs (data notshown). Surprisingly, a slightly elevated level of IFN-γ in the CD4⁺subset (but not in CD8⁺ T cells) was observed (n=3) in theanti-CD3/anti-CD28/anti-CD137 bead and PMA/ionomycin conditions in rTILscompared to eTILs. This data suggests that rTILs are functionallycompetent cells, and show evidence of greater functional competence thateTILs.

Example 4—Comparison of REP of rTILs and eTILs from Tumor Digests

eTILs and rTILs were subjected to a rapid expansion protocol (REP) withirradiated PBMC feeders, anti-CD3 antibody (OKT3), and IL-2 for 14 days.Viability and cells counts were assessed in duplicate in 3 independenttumors (n=3). Phenotypic expression was assessed by flow cytometry.Successful initiation in mini-REP experiments was observed for bothrTILs and eTILs. The REP performance of the TILs with anti-CD3 antibodyand feeders was similar (FIG. 6A), although the rTILs surprisinglyexhibited a slightly enhanced number of cells (p<0.08), compared to theeTIL. As observed prior to REP (see Example 2), the eTILs and rTILsobtained post-REP were phenotypically distinct. Many of the phenotypicdifferences observed in the pre-REP were preserved during the REP, suchas a reduction in LAG3 and TIM3 expression in the rTIL (FIG. 6B).

Additional properties of eTILs and rTILs may be compared based on theresults of (1) deep TCR sequencing, (2) co-culture proliferation(rTIL/eTIL co-culture with cytokine mixtures) and additional functionalassays, (3) assays for transcriptional profiling (e.g., using aNanoString Technologies NCOUNTER system). TCR sequencing may assess theclonality and/or diversity of the TCR repertoire, including Vbrepertoire. Telomere length may also be assessed to compare rTILs toeTILs.

Example 5—Treatment of Human Disease with rTILs and Combinations ofrTILs and eTILs

The rTILs of the invention may be used in the treatment of cancers asdescribed herein. An overall process flow diagram for the expansion ofrTILs from a patient tumor and treatment of a patient is depicted inFIG. 7. The process allows for tailoring of the rTIL to eTIL ratio inthe TIL product infused to the patient as shown. The ratio of rTIL toeTIL may be selected by way of an affinity assay or other cell sortingassay known by persons having ordinary skill in the art based on thedifferential expression of CD69 and/or T-cell exhaustive markers inrTILs and eTILs.

In FIG. 8, a timeline showing an exemplary process of obtaining rTILsfrom a patient tumor, expanding the rTILs from tumor remnants afterpre-REP using a REP stage, performing lymphodepletion, and infusion ofrTILs into a patient is shown in conjunction with a parallel eTILprocess.

Example 6—Study to Assess the VP Repertoire in eTIL and rTIL

The eTIL and rTIL were assessed for differences in the VP T cellreceptor repertoire, with respect to diversity and frequency.

In the study, 6 pre-REP eTIL/rTIL pairs were harvested from thefollowing histologies: ovarian cancer; renal cancer (n=2); and breastcancer (TNBC n=2, ER+PR+n=1). The cell pellets were shipped on dry iceto iRepertoire (Huntsville, Ala., USA) for RNA extraction and Vβsequencing.

The results of this study are illustrated in FIGS. 9-10 and 14-16. Inparticular, FIGS. 9 and 10 illustrate the diversity score and the % ofshared CDR3s, respectively. Furthermore, three clonotype graphs showingthe top shared 50 CDR3s are shown in FIGS. 14, 15, and 16 for ovariancarcinoma, renal carcinoma, and triple negative breast carcinoma,respectively.

Surprisingly, the diversity of the TCRvβ repertoire is greater in therTIL than in the eTIL (FIG. 9). Approximately 30-50% of the total CDR3in the eTIL and rTIL are shared (FIG. 10), demonstrating that a largepercentage of the total CDR3's are differentially expressed in the twopopulations. However, of the shared CDR3s the top 50 clones were mostlyshared between the two populations, suggesting that eTIL and rTIL haveclones with similar antigen specificity (see FIGS. 14-16). Moreover, thefrequency of the top 50 clones varied, suggesting again that the eTILand rTIL are surprisingly distinct T cell populations.

Example 7—Study of Co-Culture Proliferation Assays

The eTIL and rTIL were assessed determine whether the rTIL can alter theproliferation status of the eTIL, upon co-culture (or vice versa).

In the study, 5 pre-REP eTIL/rTIL pairs were harvested from thefollowing histologies: renal cancer, triple-negative breast cancer(TNBC), melanoma, lung cancer, and colorectal cancer. The rTIL wereisolated from the tumor remnants by a 60-min enzymatic digestion at 37°C. eTIL were stained with Cell Trace Yellow and rTIL with Cell Trace Redto independently track the two distinct populations. 1e6 of eTIL, 5e5eTIL+5e5 rTIL, and 1e6 rTIL were cultured for 4 days at 37° C. withIL-2+/− and OKT3 (anti-CD3 antibody) and assessed for proliferation byflow cytometry.

The results of this study are illustrated in FIG. 11. In FIG. 11, theeTIL from either the CD4+ or CD8+ population in all five tumorsdemonstrated an enhancement in the proliferative capacity uponco-culture with rTIL with anti-CD3 antibody as demonstrated by a shift(or dye dilution) in the Cell Trace dye, when compared to eTIL alone.The red represents the eTIL and the blue represents the eTIL whenco-cultured with the rTIL.

Example 8—Study of Co-Culture Proliferation Assays

The eTIL and rTIL were assessed identify similarities and/or differencesin the gene expression profile of rTIL and eTIL.

In the study, Nanostring's nCounter technology was utilized, whichemploys a color-coded barcode multiplexed to mRNA to deliver a digitalreadout of gene expression. Purified RNA (RNeasy, Qiagen) from sixmatched eTIL and rTIL samples were hybridized with an nCounterImmunology V2 panel codeset for 16 hours on a thermocycler. Codesetsconsist of a mixture of capture and reporter probes that are multiplexedwith the target RNA through 22 bp interactions during thermocycling.Samples were loaded into a 12-well SPRINT cartridge and ran on annCounter SPRINT device. Count data are exported in a custom RCC formatand matched to an RLF file which matches gene names to probe IDs.Normalization and analysis were done on nSolver 3.0 (NanoStringTechnologies, Inc.).

The results of this study are illustrated in FIGS. 12 and 13. As shownin FIGS. 12 and 13, the gene expression profile is significantlydifferent when comparing the eTIL and rTIL (see the heat map in FIG.12). There are several genes that are significantly upregulated ordownregulated in the rTIL compared to the eTIL (FIG. 13).

1. A method of treating a cancer in a patient in need of such treatment,the method comprising: administering a therapeutically effective amountof remnant tumor infiltrating lymphocytes (rTILs) to the patient,wherein the rTILs are obtained by a method comprising: (a) obtainingtumor tissue from the patient, wherein the tumor tissue comprises tumorinfiltrating lymphocytes (TILs); (b) fragmenting the tumor tissue; (c)treating the tumor tissue in a gas permeable container with a first cellculture medium and interleukin 2 (IL-2) to provide tumor remnants andemergent TILs (eTILs); (d) removing at least a plurality of the eTILs;(e) enzymatically digesting the tumor remnants into tumor remnant cellsusing a digest mixture; and (f) expanding the tumor remnant cells with asecond cell culture medium comprising cell culture media, irradiatedfeeder cells, OKT-3 antibody, and IL-2 in a gas permeable container toprovide an expanded number of the rTILs for administration to thepatient; and wherein the rTILs express reduced levels of a T cellexhaustion marker relative to the eTILs, and wherein the T cellexhaustion marker is selected from the group consisting of TIM3, LAG3,TIGIT, PD-1, CTLA-4, and combinations thereof.
 2. The method of claim 1,wherein the tumor tissue is selected from the group consisting ofmelanoma tumor tissue, head and neck tumor tissue, breast tumor tissue,renal tumor tissue, pancreatic tumor tissue, glioblastoma tumor tissue,lung tumor tissue, colorectal tumor tissue, sarcoma tumor tissue, triplenegative breast tumor tissue, cervical tumor tissue, ovarian tumortissue, and acute myeloid leukemia bone marrow or tumor tissue.
 3. Themethod of claim 1, wherein the irradiated feeder cells compriseirradiated allogeneic peripheral blood mononuclear cells.
 4. The methodof claim 1, wherein IL-2 is present in the second cell culture medium atan initial concentration of about 3000 IU/mL and OKT-3 antibody ispresent in the second cell culture medium at an initial concentration ofabout 30 ng/mL.
 5. The method of claim 1, wherein at least one T cellexhaustion marker in CD8⁺ and CD4⁺ T cells in the rTILs is reduced by atleast 10% relative to the eTILs.
 6. The method of claim 1, wherein the Tcell exhaustion marker is a LAG3 marker in CD8⁺ T cells, and wherein theLAG3 marker in the rTILs is reduced by at least 2-fold relative to theeTILs.
 7. The method of claim 1, wherein the T cell exhaustion marker isa TIM3 marker in CD8⁺ T cells, and wherein the LAG3 marker in the rTILsis reduced by at least 3-fold relative to the eTILs.
 8. The method ofclaim 1, wherein the T cell exhaustion marker is a TIM3 marker in CD4⁺ Tcells, and wherein the LAG3 marker in the rTILs is reduced by at least2-fold relative to the eTILs.
 9. The method of claim 1, wherein the TIM3marker and the LAG3 marker in the rTILs are undetectable by flowcytometry.
 10. The method of claim 1, wherein CD56⁺ expression in therTILs is reduced by at least 3-fold relative to CD56⁺ expression in theeTILs.
 11. The method of claim 1, wherein CD69⁺ expression in the rTILsis increased by at least 2-fold relative to CD69⁺ expression in theeTILs.
 12. The method of claim 1, wherein the digest mixture comprisesdeoxyribonuclease, collagenase, and hyaluronidase.
 13. The method ofclaim 1, wherein the first cell culture medium further comprises acytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21,and combinations thereof.
 14. The method of claim 1, wherein the secondcell culture medium further comprises a cytokine selected from the groupconsisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof. 15.The method of claim 1, wherein the rTILs are cryopreserved prior toadministration to the patient.
 16. The method of claim 15, furthercomprising the step of thawing the cryopreserved rTILs prior toadministration to the patient.
 17. The method of claim 1, wherein therTILs are substantially free of eTILs.
 18. The method of claim 1,wherein the step of expanding the tumor remnant cells with the secondcell culture medium comprises adding eTILs to the second cell culturemedium to provide a selected rTIL to eTIL ratio.
 19. The method of claim1, further comprising the step of adding eTIL to the rTIL to provide aselected rTIL to eTIL ratio.
 20. The method of claim 18, wherein theselected rTIL to eTIL ratio is at least 1% rTIL to eTIL.
 21. The methodof claim 20, wherein the selected rTIL to eTIL ratio is at most 99.9%rTIL to eTIL.
 22. The method of claim 18, wherein the selected rTIL toeTIL ratio is selected from the group consisting of about 1:99, about5:95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70,about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15,about 90:10, about 95:5, and about 99:1 rTIL to eTIL.
 23. (canceled) 24.The method of claim 1, further comprising the steps of: treating thepatient with a non-myeloablative lymphodepletion regimen prior toadministering the rTILs to the patient; and treating the patient with ahigh-dose IL-2 regimen starting on the day after administration of therTILs to the patient.
 25. The method of claim 24, wherein atherapeutically effective amount of eTILs are simultaneouslyadministered to the patient in a mixture with the rTILs.
 26. The methodof claim 24, wherein the non-myeloablative lymphodepletion regimencomprises the steps of administration of cyclophosphamide at a dose of60 mg/m²/day for two days followed by administration of fludarabine at adose of 25 mg/m²/day for five days.
 27. The method of claim 24, whereinthe high-dose IL-2 regimen comprises 600,000 or 720,000 IU/kg ofaldesleukin, or a biosimilar or variant thereof, administered as a15-minute bolus intravenous infusion every eight hours until tolerance.28. The method of claim 24, wherein the cancer is selected from thegroup consisting of melanoma, double-refractory melanoma, uvealmelanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer,breast cancer, head and neck cancer, renal cell carcinoma, acute myeloidleukemia, colorectal cancer, and sarcoma.
 29. The method of claim 28,wherein the cancer is breast cancer, and wherein the breast cancer isselected from the group consisting of triple negative breast cancer,estrogen receptor-positive breast cancer, progesterone receptor-positivebreast cancer, and estrogen receptor-positive/progesteronereceptor-positive breast cancer.
 30. The method of claim 28, wherein thecancer is lung cancer, and the lung cancer is selected from the groupconsisting of non-small cell lung cancer and small cell lung cancer. 31.The method of claim 1, wherein a therapeutically effective amount ofeTILs are simultaneously administered to the patient in a mixture withthe rTILs.